study of rabies virus by differential scanning calorimetry
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Study of rabies virus by Differential Scanning Calorimetry (DSC)
Frédéric Gréco Analytical Research & Developpement Biochemical & Biophysical Characterization 26 September 2016
Frédéric Gréco - DSC rabies virus
The rabies virus (RABV) continues to be a worldwide health problem.
● Rabies virus is a neurotropic virus and causes one of the most lethal zoonotic diseases – 100% of mortality in unvaccinated patients.
● Over 120 countries are still affected by canine rabies which kills someone every 9 minutes, mostly ages <15 years
● Over 55,000 people die of the disease annually, but this is probably a severe underestimation (2010)
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Worldwide distribution of RABV Human deaths in 2004 in 2007 in South-East-Asia
Scnell M. J. et al. (2010) The cell biology of rabies virus : using stealth to reach the brain, Nature Reviews Microbioogy, 8 : 51-61
Rabies virus
● Rhabdoviridae family ● Single-stranded RNA genome of about 12 kb which encodes five proteins
• the nucleoprotein (N) • the viral RNA polymerase (L) • the phosphoprotein (P) • the matrix protein (M) • The lipid bilayer • the glycoprotein (G)
● Cryo-electron microscopy image of a frozen hydrated sample of RABV
bullet-like morphology 180nm length, 80nm diameter
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Scnell M. J. and al. (2010) The cell biology of rabies virus : using stealth to reach the brain, Nature Reviews Microbioogy, 8 : 51-61
P. Guichard and al. Three dimensional morphology of rabies virus studied by cryo-electron tomography, Journal of Structural Biology 176 (2011) 32–40
*
G-protein
M/N-protein
membrane
viral particles nucleocapsid
G M
N L P
Glycoprotein G – The main antigen of the RABV vaccine
● Glycoprotein G is the only surface exposed viral protein
● This fusion protein (class III) is organized as a trimer anchored to the viral membrane
● There is a pH-dependent equilibrium between its pre- and post-fusion conformations
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Albertini A.A. and al. (2012) Molecular and cellular aspects of rhabdovirus entry, Viruses, 4(1) : 117-139
Bakker et al. (2005)
Viral membrane
Trimer of rabies glycoprotein G per homology with VSV-G structure
Roche et al. (2006) Science 313 p187
Antigenic site I : 226-231
Antigenic site II: 34-42 + 198-200
Antigenic site III: 330-338
Antigenic site IIIa: 342-343
Antigenic site IV: 251- 264
● Sanofi Pasteur as human vaccine manufacturer performs DSC analysis to
obtain information on protein tertiary structure and lot to lot consistency ● The inactivated rabies virus unfolds in two transitions In DSC
● Does it possible to assign viral protein to these events ? ● Can we go deeper in the G-protein structure with DSC ?
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DSC of rabies virions
three different batches of inactivated rabies virus analysed two times independently
40 60 80-20
0
20
40
60
80
Cp
(kca
l/mol
e/o C
)
Temperature (oC)
Sharpness of unfolding transitions and low SD of the Tm values : DSC is a good analytical tool to monitore vaccine production process and lot to lot consistency
61°C
71°C
● Viral sample are non inactivated or Beta-propiolactone (BPL)-inactivated bulk lots of strain Pitman-Moore
● Dialysis in PBS with 10kDa cutoff ● Protein 0,15-1,1mg/ml corresponding to 9.1010-7.1011 viral particles ● Experiment performed on a VP-Capillary auto-CAP DSC ● Scan rate 200°C/h to help avoiding aggregation or precipitation ● Data were normalized using the concentration of the G-protein :
approximately 25% of the viral proteins
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Study of rabies virus by DSC*
*A.Toinon and al. Biochemistry and Biophysics Reports 4 (2015) 329-336
Influence of Beta-propiolactone (BPL) inactivation
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Both unfolding events are entirely irreversible
Reversibility of unfolding inactivated virus
DSC of non inactivated and inactivated virus
Viral inactivation has a minor impact on protein unfolding
61-62°C 71-73°C
rescan
Identification of the G-protein transition
Bromelain cleavage of G-protein ectodomain* suppresses first unfolding event
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G
First unfolding event is primarily due to G unfolding
* Gaudin Y. et al. (1991)
Identification of the second transition
● Comparative DSC analysis of the entire inactivated rabies virus and
recombinantly produced purified matrix protein.
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inactivated virus
M protein
The similarities in the Tm between M protein and the 2nd event observed for the entire virus may suggest that this 2nd event may be primarily due to the unfolding of the M protein. Future studies are necessary to confirm this hypothesis
Tm 74°C M
Tm 71°C
Detection of conformational changes
● Lowering the pH, in order to mimic the acidic endosome, induces
minor changes in the unfolding behavior of inactivated rabies virus.
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From pre-fusion to post-fusion conformation some changes occur in the G-protein unfolding event
pH7,3 Tm1:61,4°C
pH6,3 Tm1:59,6°C
pH5,8 Tm1:62,2°C
Effect of reducing conditions on the G-protein conformation
● DSC shows large alteration in the unfolding behavior under reducing
conditions.
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Inactivated rabies virus in PBS in PBS + 5mM DTT
Reduction of G-protein disulfide bonds causes dramatic alterations to protein structure
They have significant effect on G structure
Interactions of RABV with monoclonal antibodies
G-protein specific monoclonal antibodies cause an up-shift of the first unfolding event confirming the notion that it corresponds to a biologically active G-protein conformation
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61°C to 65-68°C Virus only
+ Mab 50AD1
+ Mab D1-25
+ Mab TJU11-12
Antibodies alone Equimolar mixtures of RABV + Mab
Mab TJU11-12 Mab D1-25 50AD1
Antigenic site II site III
Jallet C et al (1999) J Virol. 73: 225 Dietzschold B et al (1990) J Virol. 64: p3087 Bakker et al. (2005) J. Virol., 79: p9062 Müller et al (2009) Plos, 3(11):e542
Conclusion
● DSC of RABV virions exhibits two thermal transitions.
● The transition at 61°C has a major contribution from unfolding of G proteins, whereas the transition at 71°C may mainly correspond to unfolding of M proteins.
● Conformational changes as well as binding of monoclonal antibodies to the protein G can be detected by DSC.
● Reduction of G-protein disulfide bonds causes dramatic decrease in stability of G proteins.
● DSC can be used as sensitive analytical tool to monitor the vaccine production process and lot to lot consistency.
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Analytical Research & Developpement Biochemical & Biophysical Characterization To Audrey Toinon, Nadège Moreno, Marie-Claire Nicolai, Fabienne Barrière and Catherine Manin & Frédéric Ronzon R&D Project Team To Françoise Guinet-Morlot These data were published in Biochemistry and Biophysics Reports 4 (2015) 329-336
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Acknowledgments
Frédéric Gréco - DSC rabies virus