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Hamamatsu University School of Medicine
Finding DNAvaccines against Mycobacterium Tuberculosis
Roi Villar VázquezDr. Shintaro Seto
Dr Masato UchijimaDr. Tsujimura
(HUSM, Japan)
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 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
Mycobacterium Tuberculosis Mtb
Hamamatsu University School of Medicine
DNA Vaccination4
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
MemCells
Cell to be transfected:• Myocyte (good expresser) –
MHC- I bad stimulation• APC- MHCII (bad expresser),
good estimulator• Myocyte with bioadjuvant• DC Th1 cells
BLymphocyte
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 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
Hamamatsu University School of Medicine
Objectives
1. Purify Genes Prepare pCI for DNA vaccine
2. Test Efficacy Prepare pET for protein production IFN-γ
IFN-γ
IFN-γ
IFN-γ
ELISA detection
pET
DCell
spleenocyte
Hamamatsu University School of Medicine
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
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).
Hamamatsu University School of Medicine
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 vitro 3
Hamamatsu University School of Medicine
Developing Plasmid Vaccines
Roi Villar VázquezDr. Seto
Dr. Uchijima(HUSM, Japan)
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)
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)
Hamamatsu University School of Medicine
Quality pBSII testing
• o/n culture Plasmid Purification[plasmid] determinationInsert Sequencing (discartion of rfpE and one sample of rv1996)
RE’s Reaction & purification of insert
RpfE
Rv1813c
Rv1996
rv1813c
rv1996
Mutation Screening
&
BLASTcomparison
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
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)
Hamamatsu University School of Medicine
2nd Colony PCR
• Culture colonies o/n• [plasmid] determination
(DO260)
• PCR skipped (no time)• Checking insert by
Restriction Eph map• Extract Plasmid
Hamamatsu University School of Medicine
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)
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
Hamamatsu University School of Medicine
Antigen Production through pET system in BL21(DF3) for
DNAvaccine TestingRoi Villar Vázquez
Dr.Seto
Dr. Tsujimura
Hamamatsu University School of Medicine
pET 28 from Novagen.
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
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
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
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.
Hamamatsu University School of Medicine
References1. Yasir. A.W. Skeiky et al. Advances in tuberculosis vaccine strategies2. 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 review5. David R. Sherman et al. 2001, Regulation of the Mycobacterium
Tuberculosis hypoxic response genen encoding a-crystallin6. 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 Response8. Camus,J.C., et.al . ,Re-annotation of the genome sequence of
Mycobacterium tuberculosis H37Rv 9. (Tufariello et al., 2004).10. Fenhalls et al., 2002;11. Rachman et al., 2006