Post on 16-Dec-2015
Nasty viruses
and Institutional Review Boards
WHO: 50 million cases/yr
Problem: Increased disease severity with sequential infections may be due to antibody dependent enhancement.
Macrophages do not have the normal dengue virus receptor. However, incomplete neutralization of virus can target virions to macrophage Fc receptors.
Jardetzky and Lamb, Nature 427:307-308 (2004)
Zhang, et al. Nature Structural Biology 10:907 (2003)
Zhang, et al. Nature Structural Biology 10:907 (2003)
Q: How can we find out where neutralizing antibodies target the viral envelope protein, so we can use just these areas in a vaccine?
A: Characterize the binding targets (epitopes) for individual human antibodies. (subquestion 1: how in the world do you get individual human antibodies???)
Immortalize B-cells with EBV and clone by serial dilution.
Screen for binding to DENV.
Characterize binding, neutralization, enhancement, and epitope mapping.
We’re going to need IRB approval…
Ethical questions:
Will people be hurt by this procedure?
How can we minimize this risk?
What will the fate of the cells be?
Different collaborating institutions:
FGCU
Tulane University
Tan Tock Seng Hospital
University of the West Indies
Different procedures.
Different approval process.
Different regulations.
Different consent forms.
Who even approves first?
Two full FGCU IRB applications with one amendment.
All samples blinded and coded.
Samples from Florida, Jamaica, and Singapore.
No adverse incidences.
Project ongoing, multiple HuMAbs isolated and characterized.
First description of HuMAbs against dengue virus.
Neutralization Assays
0 1:5000 1:1000 1:500 1:100 1:50-20.0
0.0
20.0
40.0
60.0
80.0
100.0
7B serum
DENV1
DENV2
DENV3
DENV4
7B serum dilution
% I
nh
ibit
ion
0 0.22 ug/ml
1.1 ug/ml
2.2 ug/ml
11 ug/ml
22 ug/ml-20.0
0.0
20.0
40.0
60.0
80.0
100.0
2.3D mAb
DENV1
DENV2
DENV3
DENV4
% I
nh
ibit
ion
0 0.4 ug/ml
2.0 ug/ml
4.0 ug/ml
20 ug/ml
40 ug/ml-20
0
20
40
60
80
100
3.6D mAb
DENV1
DENV2
DENV3
DENV4
% I
nh
ibit
ion
0 0.4 ug/ml
2.0 ug/ml
4.0 ug/ml
20 ug/ml
40 ug/ml-20
0
20
40
60
80
100
4.8A mAb
DENV1
DENV2
DENV3
DENV4
% I
nh
ibit
ion
Neutralizing and non-neutralizing monoclonal antibodies against dengue virus Eprotein derived from a naturally infected patient
Virology Journal 2010, 7:28 doi:10.1186/1743-422X-7-28
John S Schieffelin (jschieff@tulane.edu)Joshua M Costin (jcostin@fgcu.edu)Cindo O Nicholson (cnicholson@fgcu.edu)Nicole M Orgeron (norgeron@tulane.edu)Krystal A Fontaine (krystala@u.washington.edu)Sharon Isern (sisern@fgcu.edu)Scott F Michael (smichael@fgcu.edu)James E Robinson (jrobinso@tulane.edu)
FGCU
Sharon Isern, PhD
Joshua Costin, PhD
Kelli Barr, PhD
Yancey Hrobowski, PhD
Krystal Fontaine
Cindo Nicholson
Craig Rees
Tom Everts
Nadiya Joseph
Collaborators
Li Lin, MD - Tan Tock Seng
John Lindo, MD - UWI Mona
James Robinson, MD - Tulane
John Schefflein, MD - Tulane
Funding
US National Institutes of Health
US Department of Defense
Acknowledgements