Finding DNAvaccines against Mycobacterium Tuberculosis

1. Finding DNAvaccines against Mycobacterium Tuberculosis Roi Villar Vázquez Dr. Shintaro Seto Dr Masato Uchijima Dr. Tsujimura (HUSM, Japan)

2. Tuberculosis: Pandemian Menace  Mtb Mycobacterium Tuberculosis • 2nd Infectious death Cause (2.000.000deaths/year) (HIV) 1,6 • 2.000.000.000 infected, 8.000.000 new cases in developing countries each year1 • LTBI in hypoxia- High incidence, High expression pattern change. 5,6 • Multi-Drug Resistant Strains (Eastern Europe)1 • BCG non always functional with variable eficacy. Unsafe.6

3. DNA Vaccination4 Cytotoxic T lymphocyte Simulate a somatic Cell being infected by a pathogen. MHC I Plasmids: • Stability&Easy Storage • Cheap production • Safe administration • Non allergenic • Booster effect. Mem Cells Helper T lymphocyte B Lymphocyte Cell to be transfected: • Myocyte (good expresser) – MHC- I bad stimulation • APC- MHCII (bad expresser), good estimulator • Myocyte with bioadjuvant • DC Th1 cells

4. Gene Delivery (Gene’s Therapy)4,6 • Transfection in vitro & reimplatation • Transduction with Virus /Bacteria (unsafe) • Transfection in vivo • Gene Gun (lowest [p] needed, not high protective) • Mucosal injectors • Electroporation • Cationic/lipid microparticles Other Improvements6 • Heterologous Regime raises Booster Effect when DNAv is priming vaccine • Chimeric DNAvaccines (Fusion proteins) enhace immune response6 (eg. MIP1a DC or BP to a Imm cell recerptor, chlatrine endocytosis )7 • Co expression of chemichal immune signals

5. Objectives • Purify Genes Prepare pCI for DNA vaccine DCell • Test Efficacy Prepare pET for protein production IFN-γ IFN-γ IFN-γ IFN-γ pET ELISA detection spleenocyte

6. Vaccine Candidates:Rv1813c8 • 143 aa. • Conserved hypothetical protein. Possibly a exported protein with potential N-terminal signal sequence. • Similar to Q11050|Rv1269c|MTCY50. • Entrez Gene: Rv1813c hypothetical protein [ Mycobacterium tuberculosis H37Rv ] GeneID: 885546

7. Vaccine Candidates: Rv1996 • Len : 317 aa. Conserved hypothetical protein • Conserved domains with Universal stress proteins and related nucleotide-binding proteins  Start of dormancy state by hypoxia • Similar to several Mycobacterium tuberculosis hypothetical proteins e.g. Rv2005c|Q10851|YK05_MYCTU (295 aa), FASTA scores: opt: 775, E(): 0, (50.3% identity in 316 aa overlap); Rv2026c (294 aa) (47.9% identity in 311 aa overlap); and Rv2623, etc. Also similar to SCJ1.30c|AL109962 hypothetical protein from Streptomyces coelicolor (328 aa).

8. Vaccine Candidates: rpfE • len: 172 aa, possible secretory signal sequence in N-terminus. • Secreted3 lytic transglycosylases of mycobacteria, known as resuscitation-promoting E (RpfE) 2 • expressed in vitro and in mice9 also been observed in human TB infection10,11 (Fenhalls et al., 2002; Rachman et al., 2006). • Though not formally considered virulence factors, genes required for bacterial cell division clearly are necessary for the growth, and thus, pathogenesis, of bacteria. 2Rpf proteins constitute a family of lytic transglycosylase enzymes capable of hydrolyzing the glycosidic bonds in the essential stress-bearing, shape-maintaining peptidoglycan layer 2 • The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro3

9. Developing Plasmid Vaccines Roi Villar Vázquez Dr. Seto Dr. Uchijima (HUSM, Japan)

10. 1.Colony PCR • 3 Ecoli plates transformed with plasmid ( pBSII) (Blue&white colonies). Containing RT-PCR cDNA product • Select 6 white colonies from each plate, Re-culture on a plate. Name them 22 (rfpE) 23 (rv1813c) 24 (rv1996)

11. 1.Colony PCR • Check if pBSII’s replicas have our insert • Amplify Insert with P7&P8 primers • Check existance of insert by Agarose Gel Electrophoresis • Choose colonies. • Culture o/n 22 (rfpE) 23 (rv1813c) • Rv1996 w/o criteria. No amplified insert seen. 24 (rv1996)

12. Quality pBSII testing • o/n culture  Plasmid Purification • [plasmid] determination • Insert Sequencing (discartion of rfpE and one sample of rv1996) • RE’s Reaction & purification of insert Mutation Screening & BLAST comparison RpfE Rv1813c rv1813c Rv1996 rv1996

13. pCI Cloning & DNAv. • Cut both plasmid donor & receptor. Separate with Agar eph • Purify insert and open pCI, ligate them, transformate HS & culture o/n (Ap) Rv1813c MiuI &XhoI Rv1996:EcoRI &KpnI

14. 2nd Colony PCR • 2 Ecoli plates transformed with pCI-gene • Select 4 white colonies from each plate, Re-culture on a plate. Name them pCI-(rv1813c) pCI-(rv1996)

15. 2nd Colony PCR • Culture colonies o/n • [plasmid] determination (DO260) • PCR skipped (no time) • Checking insert by Restriction Eph map • Extract Plasmid

16. Large Preparation of pCI • New transformation of E.coli • Large Plasmid Extraction • [plasmid] determination DO260 • Sequencing insert in pCI (control of contamination between samples) Unsuccessful (high annealing Tª)

17. Administration of DNA vaccine • Preparation of Coated Gold Particles As Gene Gun Protocol. • Plasmid transfection with Gene Gun. • Homologous regime vaccination  Another transfection must be made in 2 weeks. • ELISA test for testing immunisation

18. Antigen Production through pET system in BL21(DF3) for DNAvaccine Testing Roi Villar Vázquez Dr.Seto Dr. Tsujimura

19. pET 28 from Novagen.

20. Cloning into pET • Amplification of pET by transformation (HS), o/n culture, and pET extraction • [plasmid] determination. • Study of framing Restriction Sites. • Restriction Rx. (Also pET) • pBSII-rv1813 – cut w/ HindIII & NotI • pBSII-rv1996 – cut w/ EcoRI * • SAP treatment on pET • Electrophoresis on Agarose (4 samples) • Ligation, TransformationHS and Culture o/n Kn

21. Quality Test & Protein production • Restriction Map to test insertion / insert orientation rv1996 discarted • Transformation rv1813c in BL21(DE3) & culture o/n. • Pick 3 colonies, culture them and divide in 3 tubes each: master, (+) control w/ IPTG, (-) control w/o IPTG. • SDS PAGE of lysates of 2nd &3rd: No clear Result

22. Protein Production • 5ml Culture from 3 Master tubes • Induction with IPTG. • Extraction with Ni- NTA column under denaturant conditions (Urea 8M) Mw C W E C W E C W E • SDS- PAGE of • Crude • Washed • Eluate • No clear results.

23. Comments & Conclusions. • rfpE should be cloned and tested again (just sequencing rx was wrong). • Rv1996 and & Rv1813c, should be reinserted into pET, avoiding SAP dangerous treatment. Using pET-28 a or c to avoid frame shifting. Cloning them from pCI. • pCI should codify some bio-adjuvant to enhance immune response as Ag is synthetised alone.

24. References • Yasir. A.W. Skeiky et al. Advances in tuberculosis vaccine strategies • A Mycobacterial Enzyme Essential for Cell Division Synergizes with Resuscitation-Promoting Factor:Erik C. Hett et al. • The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro Bavesh D Kana, et al • M.A. Liu 2003, DNA vaccines: a review • David R. Sherman et al. 2001, Regulation of the Mycobacterium Tuberculosis hypoxic response genen encoding a-crystallin • Umesh Datta Gupta et al, 2007 Current Status of TB Vaccines ( vaccine) • M. Uchijima et al (2008), chemokine receptor mediated delivery of mycobacterium MPT-51 protein induces Antigen specific Tcell Response • Camus,J.C., et.al . ,Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv • (Tufariello et al., 2004). • Fenhalls et al., 2002; • Rachman et al., 2006