Nasty
viruses
WHO: 50 million cases
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
7B serum
2.3D mAb
80.0
80.0
60.0
DENV1
DENV2
40.0
DENV3
20.0
DENV4
0.0
0
1:5000 1:1000 1:500 1:100
% Inhibition
100.0
% Inhibition
100.0
1:50
60.0
DENV1
DENV2
40.0
DENV3
DENV4
20.0
0.0
0
-20.0
7B serum dilution
-20.0
4.8A mAb
100
100
80
80
DENV1
DENV2
40
DENV3
DENV4
20
60
DENV1
DENV2
40
DENV3
DENV4
20
0
0
0
-20
% Inhibition
% Inhibition
3.6D mAb
60
0.22
1.1
2.2
11
22
ug/ml ug/ml ug/ml ug/ml ug/ml
0.4
2.0
4.0
20
40
ug/ml ug/ml ug/ml ug/ml ug/ml
0
-20
0.4
2.0
4.0
20
40
ug/ml ug/ml ug/ml ug/ml ug/ml
Neutralizing and non-neutralizing monoclonal
antibodies against dengue virus E
protein 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)
Acknowledgements
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