Outline

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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.

10. Genes cloned into master vector using Gateway Method • Recombination sites added to ORF by PCR primers. • PCR products screened on gels and by sequencing.

11. 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.

13. 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.

14. 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.

15. Gateway Method

16. 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.

17. 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

18. 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.

19. 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.

20. Vectors -VR1020 DNA Vaccine Vector Contains: • Ampicillin • ccdB gene • recombination sites. • Expression driven by CMV promoter- viral • tPA leader sequence

21. 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

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

23. 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

24. Inactive Attenuated (live) Toxoids Subunits Conjugated Recombinant DNA Vaccine Avian Flu developed by reverse genetics Vaccines

25. P. Falciparum and P. Yoelii both have genomes sequenced Translates into reverse vaccinology Chromosome 3 of P. falciparum Why Reverse Genetics?

26. What Constructs Were Tested ORF’s thought to code for membrane proteins

27. 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.

28. 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

29. 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