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

2. Hamamatsu University School of Medicine Tuberculosis: Pandemian Menace 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 year 1 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 Mycobacterium Tuberculosis  Mtb

3. Hamamatsu University School of Medicine DNA Vaccination 4 Plasmids: Stability&Easy Storage Cheap production Safe administration Non allergenic Booster effect. Simulate a somatic Cell being infected by a pathogen. MHC I Cytotoxic T lymphocyte Helper T lymphocyte Mem Cells Cell to be transfected: Myocyte (good expresser) – MHC- I bad stimulation APC- MHCII (bad expresser), good estimulator Myocyte with bioadjuvant DC  Th1 cells B Lymphocyte

4. Hamamatsu University School of Medicine 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 Improvements 6 Heterologous Regime raises Booster Effect when DNAv is priming vaccine Chimeric DNAvaccines (Fusion proteins) enhace immune response 6 (eg. MIP1a  DC or BP to a Imm cell recerptor, chlatrine endocytosis ) 7 Co expression of chemichal immune signals

5. Hamamatsu University School of Medicine Objectives 1.Purify Genes  Prepare pCI for DNA vaccine 2.Test Efficacy  Prepare pET for protein production IFN-γ ELISA detection pET DCell spleenocyte

6. Hamamatsu University School of Medicine Vaccine Candidates:Rv1813c 8 143 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. Hamamatsu University School of Medicine 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. Hamamatsu University School of Medicine Vaccine Candidates: rpfE len: 172 aa, possible secretory signal sequence in N-terminus. Secreted 3 lytic transglycosylases of mycobacteria, known as resuscitation- promoting E (RpfE) 2 expressed in vitro and in mice 9 also been observed in human TB infection 10,11 (Fenhalls et al., 2002; Rachman et al., 2006).Fenhalls et al., 2002Rachman 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. 2 Rpf 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 vitro 3

9. Hamamatsu University School of Medicine Developing Plasmid Vaccines Roi Villar Vázquez Dr. Seto Dr. Uchijima (HUSM, Japan)

10. Hamamatsu University School of Medicine 1.Colony PCR 1.3 Ecoli plates transformed with plasmid ( pBSII) (Blue&white colonies). Containing RT-PCR cDNA product 2.Select 6 white colonies from each plate, Re-culture on a plate. Name them 22 (rfpE) 23 (rv1813c)24 (rv1996)

11. Hamamatsu University School of Medicine 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 Rv1996 w/o criteria. No amplified insert seen. 22 (rfpE) 23 (rv1813c) 24 (rv1996)

12. Hamamatsu University School of Medicine 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 RpfE Rv1813c Rv1996 rv1813c rv1996 Mutation Screening & BLAST comparison

13. Hamamatsu University School of Medicine 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. Hamamatsu University School of Medicine 2nd Colony PCR 1.2 Ecoli plates transformed with pCI- gene 1.Select 4 white colonies from each plate, Re-culture on a plate. Name them pCI-(rv1813c)pCI-(rv1996)

15. Hamamatsu University School of Medicine 2nd Colony PCR Culture colonies o/n [plasmid] determination (DO 260 ) PCR skipped (no time) Checking insert by Restriction Eph map Extract Plasmid

16. Hamamatsu University School of Medicine Large Preparation of pCI New transformation of E.coli Large Plasmid Extraction [plasmid] determination DO 260 Sequencing insert in pCI (control of contamination between samples) Unsuccessful (high annealing Tª)

17. Hamamatsu University School of Medicine 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. Hamamatsu University School of Medicine Antigen Production through pET system in BL21(DF3) for DNAvaccine Testing Roi Villar Vázquez Dr.Seto Dr. Tsujimura

19. Hamamatsu University School of Medicine pET 28 from Novagen.

20. Hamamatsu University School of Medicine Cloning into pET 1.Amplification of pET by transformation (HS), o/n culture, and pET extraction 2.[plasmid] determination. 3.Study of framing Restriction Sites. 4.Restriction Rx. ( Also pET) –pBSII-rv1813 – cut w/ HindIII & NotI –pBSII-rv1996 – cut w/ EcoRI *  SAP treatment on pET 5.Electrophoresis on Agarose (4 samples) 6.Ligation, TransformationHS and Culture o/n Kn

21. Hamamatsu University School of Medicine Quality Test & Protein production 1.Restriction Map to test insertion / insert orientation rv1996 discarted 2.Transformation rv1813c in BL21(DE3) & culture o/n. 3. Pick 3 colonies, culture them and divide in 3 tubes each: master, (+) control w/ IPTG, (-) control w/o IPTG. 4. SDS PAGE of lysates of 2nd &3rd: No clear Result

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

23. Hamamatsu University School of Medicine 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. Hamamatsu University School of Medicine References 1.Yasir. A.W. Skeiky et al. Advances in tuberculosis vaccine strategies 2.A Mycobacterial Enzyme Essential for Cell Division Synergizes with Resuscitation-Promoting Factor:Erik C. Hett et al. 3.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 4.M.A. Liu 2003, DNA vaccines: a review 5.David R. Sherman et al. 2001, Regulation of the Mycobacterium Tuberculosis hypoxic response genen encoding a-crystallin 6.Umesh Datta Gupta et al, 2007 Current Status of TB Vaccines ( vaccine) 7.M. Uchijima et al (2008), chemokine receptor mediated delivery of mycobacterium MPT-51 protein induces Antigen specific Tcell Response 8.Camus,J.C., et.al.,Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv 9.(Tufariello et al., 2004).Tufariello et al., 2004 10.Fenhalls et al., 2002;Fenhalls et al., 2002 11. Rachman et al., 2006Rachman et al., 2006