Outline

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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

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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

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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

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Plasmodium falciparum

• Genome sequenced in 2002
• whole chromosome shotgun
• 14 chromosomes
• 23-megabase genome
• 5,300 protein-encoding genes
• highest (AT) 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

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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 AT content causes high frequencies of
  nonrecombinant or rearranged clones

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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
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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

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• 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.

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Genes cloned into master vector using Gateway
Method

• Recombination sites added to ORF by PCR primers.
• PCR products screened on gels and by sequencing.

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• 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.

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• 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 ? intergrase recombination
  proteins for directional recombination.

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• 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.
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Gateway Method
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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.

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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

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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.

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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.

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Vectors -VR1020 DNA Vaccine Vector

• Contains
• Ampicillin
• ccdB gene
• recombination sites.
• Expression driven by CMV promoter- viral
• tPA leader sequence

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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

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Immune System

• 1st Tier Physical Barriers
• 2nd Tier Innate Immune System
• 3rd Tier Adaptive Immune System

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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

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Vaccines

• Inactive
• Attenuated (live)
• Toxoids
• Subunits
• Conjugated
• Recombinant
• DNA Vaccine

• Avian Flu developed by reverse genetics

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Why Reverse Genetics?

• P. Falciparum and P. Yoelii both have genomes
  sequenced
• Translates into reverse vaccinology

• Chromosome 3 of
• P. falciparum

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What Constructs Were Tested

• ORFs thought to code for membrane proteins

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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.

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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

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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