hamamatsu university school of medicine finding dnavaccines against mycobacterium tuberculosis roi...
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![Page 1: Hamamatsu University School of Medicine Finding DNAvaccines against Mycobacterium Tuberculosis Roi Villar Vázquez Dr. Shintaro Seto Dr Masato Uchijima](https://reader030.vdocuments.site/reader030/viewer/2022032606/56649eb05503460f94bb56e6/html5/thumbnails/1.jpg)
Hamamatsu University School of Medicine
Finding DNAvaccines against Mycobacterium Tuberculosis
Roi Villar VázquezDr. Shintaro Seto
Dr Masato UchijimaDr. Tsujimura
(HUSM, Japan)
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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
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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
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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
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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
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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
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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).
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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
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Hamamatsu University School of Medicine
Developing Plasmid Vaccines
Roi Villar VázquezDr. Seto
Dr. Uchijima(HUSM, Japan)
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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)
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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)
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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
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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
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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)
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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
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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)
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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
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Hamamatsu University School of Medicine
Antigen Production through pET system in BL21(DF3) for
DNAvaccine TestingRoi Villar Vázquez
Dr.Seto
Dr. Tsujimura
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Hamamatsu University School of Medicine
pET 28 from Novagen.
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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
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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
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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
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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.
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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