Post on 23-Feb-2016
description
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
DENV1DENV2DENV3DENV4
7B serum dilution
% In
hibi
tion
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
DENV1DENV2DENV3DENV4
% In
hibi
tion
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
DENV1DENV2DENV3DENV4
% In
hibi
tion
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
DENV1DENV2DENV3DENV4
% In
hibi
tion
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)
FGCUSharon Isern, PhD
Joshua Costin, PhDKelli Barr, PhD
Yancey Hrobowski, PhDKrystal FontaineCindo Nicholson
Craig ReesTom Everts
Nadiya Joseph
CollaboratorsLi Lin, MD - Tan Tock Seng
John Lindo, MD - UWI MonaJames Robinson, MD - TulaneJohn Schefflein, MD - Tulane
FundingUS National Institutes of Health
US Department of Defense
Acknowledgements