Craigo, JK

Gearhart, TL

Montelaro, RC

Steckbeck, JD

Retrovirology

PubMed

TL Gearhart

JD Steckbeck

JK Craigo

RC Montelaro

Springer - Publisher Connector

POSTER PRESENTATION Open Access
Rate and affinity binding constants determined
by SPR spectroscopy reveal differential
antigenicity of HIV gp120 and gp140
TL Gearhart*, JD Steckbeck, JK Craigo, RC Montelaro
From AIDS Vaccine 2012
Boston, MA, USA. 9-12 September 2012
Background
The development of a practical, effective AIDS vaccine
that can produce enduring broadly protective immunity to
natural exposure by diverse strains of HIV-1 remains the
ultimate goal for controlling the ongoing AIDS epidemic.
The success of an effective AIDS vaccine will likely depend
on designing immunogens that elicit broadly neutralizing
antibodies to circulating HIV-1 strains, and it is important
to thoroughly understand the mechanism of binding of
HIV antigens and neutralizing antibodies. The current
study was designed to determine differences in the specifi-
city and quantitative properties of antibody binding to
gp120 or gp140 envelope proteins derived from natural
HIV isolates.
Methods
We previously described novel assays of Env-specific
serum antibodies elicited by animal lentiviral and HIV
infections and immunizations that measure qualitative
properties of avidity and conformational dependence in an
ELISA format. However, recent data have emphasized lim-
itations of this format. To provide a more sensitive, speci-
fic, and reproducible format for our antibody assays, we
have recently developed novel procedures using SPR spec-
troscopy as measured in the Biacore system to characterize
real time reaction kinetics (association/dissociation rates).
Results
The assays conducted under physiological conditions
(37°C, nondenaturing conditions, etc.) clearly indicate
the potential of these kinetic measurements of antigen-
antibody binding to provide novel data not achieved
with standard assays. We have used these binding assays
and a panel of well-characterized monoclonal antibodies
both to linear and conformational binding domains to
characterize differences in reactivity between variant
gp120 and gp140 antigens as determined by kinetic
rates and affinity of antibody binding. We have identi-
fied significant distinguishing differences in several bind-
ing characteristics of monoclonal antibody binding to
gp120 vs gp140 antigens.
Conclusion
These kinetic parameters offer novel insights into the
fundamental interactions of reference monoclonal anti-
bodies and variant Env proteins and have the potential
to provide novel correlates of neutralization in vitro and
protection in vivo.
Published: 13 September 2012
doi:10.1186/1742-4690-9-S2-P59
Cite this article as: Gearhart et al.: Rate and affinity binding constants
determined by SPR spectroscopy reveal differential antigenicity of HIV
gp120 and gp140. Retrovirology 2012 9(Suppl 2):P59.
University of PIttsburgh, Pittsburgh, PA, USA
Gearhart et al. Retrovirology 2012, 9(Suppl 2):P59
http://www.retrovirology.com/content/9/S2/P59
© 2012 Gearhart et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.


Rate and affinity binding constants determined by SPR spectroscopy reveal differential antigenicity of HIV gp120 and gp140

D-Scholarship@Pitt

POSTER PRESENTATION Open AccessRate and affinity binding constants determinedby SPR spectroscopy reveal differentialantigenicity of HIV gp120 and gp140TL Gearhart*, JD Steckbeck, JK Craigo, RC MontelaroFrom AIDS Vaccine 2012Boston, MA, USA. 9-12 September 2012BackgroundThe development of a practical, effective AIDS vaccinethat can produce enduring broadly protective immunity tonatural exposure by diverse strains of HIV-1 remains theultimate goal for controlling the ongoing AIDS epidemic.The success of an effective AIDS vaccine will likely dependon designing immunogens that elicit broadly neutralizingantibodies to circulating HIV-1 strains, and it is importantto thoroughly understand the mechanism of binding ofHIV antigens and neutralizing antibodies. The currentstudy was designed to determine differences in the specifi-city and quantitative properties of antibody binding togp120 or gp140 envelope proteins derived from naturalHIV isolates.MethodsWe previously described novel assays of Env-specificserum antibodies elicited by animal lentiviral and HIVinfections and immunizations that measure qualitativeproperties of avidity and conformational dependence in anELISA format. However, recent data have emphasized lim-itations of this format. To provide a more sensitive, speci-fic, and reproducible format for our antibody assays, wehave recently developed novel procedures using SPR spec-troscopy as measured in the Biacore system to characterizereal time reaction kinetics (association/dissociation rates).ResultsThe assays conducted under physiological conditions(37°C, nondenaturing conditions, etc.) clearly indicatethe potential of these kinetic measurements of antigen-antibody binding to provide novel data not achievedwith standard assays. We have used these binding assaysand a panel of well-characterized monoclonal antibodiesboth to linear and conformational binding domains tocharacterize differences in reactivity between variantgp120 and gp140 antigens as determined by kineticrates and affinity of antibody binding. We have identi-fied significant distinguishing differences in several bind-ing characteristics of monoclonal antibody binding togp120 vs gp140 antigens.ConclusionThese kinetic parameters offer novel insights into thefundamental interactions of reference monoclonal anti-bodies and variant Env proteins and have the potentialto provide novel correlates of neutralization in vitro andprotection in vivo.Published: 13 September 2012doi:10.1186/1742-4690-9-S2-P59Cite this article as: Gearhart et al.: Rate and affinity binding constantsdetermined by SPR spectroscopy reveal differential antigenicity of HIVgp120 and gp140. Retrovirology 2012 9(Suppl 2):P59.University of PIttsburgh, Pittsburgh, PA, USAGearhart et al. Retrovirology 2012, 9(Suppl 2):P59http://www.retrovirology.com/content/9/S2/P59© 2012 Gearhart et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

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Rate and affinity binding constants determined by SPR spectroscopy reveal differential antigenicity of HIV gp120 and gp140

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