Transcript Slide 1

Outline
• Background on Malaria and
Plasmodium life cycle.
• Gateway cloning and using Expression
vectors.
• Immune system, reverse vaccinology, and
antibody study in mice.
• Future Studies
Malaria
• Vector-borne infectious disease, caused by Plasmodium parasites
–P. falciparum – most lethal
–P. vivax
–P. ovale
–P. malariae
• Transmitted by female Anopheles mosquitoes
• 300-500 million infected per year, 1-3 million deaths per year
• Children under 5 years and pregnant women at higher risk due to
decreased immune response
Malaria Life Cycle
• In the liver…
–sporozoite invades liver
cell
–produces 30,000-40,000
daughter merozoite cells
in 6 days
–liver cell ruptures and
releases merozoites
• In the blood…
–merozoite invades red
blood cell
–produces 8-24 daughter
merozoite cells in 48 hrs
–red blood cell ruptures
and releases merozoites
• To avoid infected red blood cell detection/destruction by the spleen, Plasmodium
display the protein PfEMP1 on the surface of the red blood cells causing the red
blood cell to adhere to blood vessel walls
Plasmodium falciparum
• Genome sequenced in 2002
– whole chromosome shotgun
– 14 chromosomes
– 23-megabase genome
– 5,300 protein-encoding genes
– highest (A+T) rich sequence to date
76.3% exons, 86.5% introns
and intergenic regions, 80.6%
overall
• Paper… most genes examined
are highly expressed in sporozoite
stage
Challenges in Developing a Vaccine Against
Malaria
• Complex multi-stage Plasmodium life cycle
• Large genome size and number of protein-encoding genes
• Distinct stage-specific gene expressions require distinct immune
response targets for each stage
• High A+T content causes high frequencies of nonrecombinant or
rearranged clones
Reverse Vaccinology
• Conventional methods
– grow microbes in vitro
– harvest microbial proteins that may have the
potential to produce a vaccine
– problem… many microbes are hard to cultivate
in vitro
• Reverse vaccinology methods
– computer analyzes specific microbe genome
sequence to predict genes that code for surface
proteins
– computers can examine multiple microbial
genomes to look for homologous proteins to
create more accurate predictions
– predicted genes are cloned into E. coli cultures
for manufacturing of recombinant proteins – these
are the potential vaccines
– proteins are extracted from the E. coli cultures
and tested in mice for the immune response of
interest
Schematic representation of the essential steps of vaccine development by the conventional approach and by reverse vaccinology. Rino Rappuoli Current
Opinion in Microbiology, Volume 3, Issue 5, 1 October 2000, Pages 445-450
Outline
• Background on Malaria and Plasmodium
life cycle.
• Gateway cloning and using Expression
vectors.
• Immune system, reverse vaccinology, and
antibody study in mice.
• Future Studies
303 target genes were selected to be cloned.
• Of these, 111 single exon ORF were chosen
from chromosome 2 and 3. (prior to full
sequencing of genome).
Size range:(300-3000bp)
• 192 both spliced and single exon ORFs were
selecting using bioinformatics and comparative
genomics tools.
Size range:(200-5500bp)
• Genes chosen were found to have been
expressed in sporozites by various methods.
Genes cloned into master vector
using Gateway Method
• Recombination sites added to ORF by PCR
primers.
• PCR products screened on gels and by
sequencing.
•
Figure 2 PCR screening BP
and LR reactions. (A)
Recombinant clones in the
pDONR/Zeo master plasmid
were screened by colony
PCR using plasmid-specific
primers (M13 forward and
reverse). The figure shows
amplified DNA products from
12 BP reactions representing
different genes in groups of
four colonies (a, b, c, d) per
clone analyzed on a 1%
agarose gel. (B) The
screening of recombinants
clones in the VR1020-DV
destination vector was done
by PCR on DNA from 4 single
colonies (a, b, c, d) as well as
bulk cultures (B) from the
same transformation. Clones
shown were picked at
random. (M) 1-kb DNA
extension ladder.
From Master clones ORF can be sub cloned
into Protein expression vectors and DNA
vaccine vectors.
This uses “recombinational cloning” or the
gateway method instead of traditional
restriction digest and ligation into vectors.
Recombinational cloning is highly efficient
and uses bacteriophage λ intergrase
recombination proteins for directional
recombination.
Genes flanked by
recombination sites
can be mixed in vitro
with intergrase
proteins to transfer the
gene to a new vector.
To isolate the Destination
vector the mixture is
introduced to E.coli by
transformation and selected
for.
Gateway Method
The vector of interest is selected
for two ways:
• The master clone contains kanamycin
resistance genes. While the expression
vectors contain Ampicillin resistance
genes.
• The expression vector contains a selection
marker (F-plasmid-encoded ccdB gene)
which is replaced by the gene of interest
when recombination occurs.
• ccdB is an inhibitor of cell growth.
Vectors - PDEST17
E. coli Expression Vector
Makes fusion proteins containing
six histidines at the N terminus.
Contains:
• Ampicillin
• ccdB gene
• recombination sites.
• T7 RNA pol promoter
• Controlled by salt-inducible
promoter
Vectors – pMAL-2c
E. coli Expression
Vector
Upstream malEgene (maltose
binding protein MBP). Results
in a MBP fusion protein.
Vector Contains:
• Ampicillin
• ccdB gene
• recombination sites.
• Ptac promoter
Protein of interest can be easily
purified and isolated from MBP.
Protein Expression Results
• Although expression studies using
gateway vectors have been successful
using human ORFs. Parliamentary
attempts to express Plasmodium ORF was
not successful.
– 7 out of 95 fusion proteins were expressed
This may be due to toxicity of the
expressed genes to E. coli or to the
relative size of the expressed genes to
those not expressed successfully.
Vectors -VR1020
DNA Vaccine Vector
Contains:
• Ampicillin
• ccdB gene
• recombination sites.
• Expression driven by CMV
promoter- viral
• tPA leader sequence
Outline
• Background on Malaria and Plasmodium
life cycle.
• Gateway cloning and using Expression
vectors.
• Immune system, reverse vaccinology,
and antibody study in mice.
• Future Studies
Immune System
• 1st Tier: Physical
Barriers
• 2nd Tier: Innate
Immune System
• 3rd Tier: Adaptive
Immune System
Antibodies
• Main function of the
humoral immune
system
• Exists freely in the
blood and on cell
membranes
• Tags the antigen for
destruction by other
parts of the immune
system
Vaccines
•
•
•
•
•
•
•
Inactive
Attenuated (live)
Toxoids
Subunits
Conjugated
Recombinant
DNA Vaccine
Avian Flu developed by
reverse genetics
Why Reverse Genetics?
• P. Falciparum and P.
Yoelii both have
genomes sequenced
• Translates into
reverse vaccinology
Chromosome 3 of
P. falciparum
What Constructs Were Tested
ORF’s thought to
code for membrane
proteins
Results
• IFAT
Indirect
immunofluorescence
assay: A laboratory test
used to detect antibodies
in serum or other body
fluid. The specific
antibodies are labeled
with a compound that
makes them glow an
apple-green color when
observed microscopically
under ultraviolet light.
Analysis
• A majority of these genes induced antibodies
that reacted with erythrocytic stages
• most (17/19) of the positive genes had high
expression levels during the erythrocytic stages
of development, both at RNA and protein profiles
• The remaining 74 DNA constructs that failed to
induce antibodies to parasites generally were
not identified in the proteome analysis in the
parasite stages evaluated; only 24 of these
genes had peptides detected from any parasite
stage examined
Research Possibilities using the Gateway
System
• Generate high-throughput expression vectors
for…
– protein microarray experiments and animal immunization
– large-scale transfection experiments to assay protein
localization
– determining immunogenicity of candidate antigens
– Y2H systems to screen for host receptors and parasite
protein interactions
– developing large numbers of recombinant virus constructs
that can be used in immune assays for screening positive
antigens
– functional identification of the large number of unknown and
hypothetical proteins in the P. falciparum genome