finding dnavaccines against mycobacterium tuberculosis

Hamamatsu University School of Medicine

Finding DNAvaccines against Mycobacterium Tuberculosis

Roi Villar VázquezDr. Shintaro Seto

Dr Masato UchijimaDr. Tsujimura

(HUSM, Japan)


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


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


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


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


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


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).


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


Developing Plasmid Vaccines

Roi Villar VázquezDr. Seto

Dr. Uchijima(HUSM, Japan)


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)


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)


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


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


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)


2nd Colony PCR

• Culture colonies o/n• [plasmid] determination

(DO260)

• PCR skipped (no time)• Checking insert by

Restriction Eph map• Extract Plasmid


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)


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


Antigen Production through pET system in BL21(DF3) for

DNAvaccine TestingRoi Villar Vázquez

Dr.Seto

Dr. Tsujimura


pET 28 from Novagen.


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


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


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


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.


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

finding dnavaccines against mycobacterium tuberculosis

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