Oral Abstract Session 01 - Global HIV Vaccine Enterprise

POSTERS
Posters
Topic 3: B Cell Immunology and Antibody Functions
P03.15
The Impact of Differences in Viral Entry and
Trafficking on the Mechanism of HIV-1 Neutralization
C.D. Macedo Cincotta 1 , L.M. Wieczorek 1 , B. Barrows 1 ,
B. Brown 2 , L. Asher 1 , N. Michael 3 , M. Marovich 3 , V.R. Polonis 3
1 Henry M Jackson Foundation/ U.S. Military HIV Research
Program (MHRP), Silver Spring, MD, USA; 2 Termofisher, USA;
3 Military HIV Research Program (MHRP)/ WRAIR, USA
Background: Cellular entry of HIV-1 occurs through fusion at the
plasma membrane (PM) or via endocytosis. PM fusion, resulting in
deposit of nucleocapsid into the cytoplasm, typically establishes
productive infection. In contrast, entry through endocytosis
results in either lysosomal degredation, or productive infection
through endosomal membrane fusion. While viral entry and
permissivity are known to differ between host cells, the influence
of these differences on antibody-mediate neutralization is
unknown. Here we explore the relationships between HIV-1
entry, neutralization sensitivity, and the mechanisms by which
antibodies act.
Methods: Differences in viral entry pathways between
peripheral blood mononuclear cells (PBMC) and TZM-bl cells
were visualized by electron microscopy (EM). Cells were
incubated with HIV, with or without antibody, for 1 hour at
4oC, then viral entry was allowed for 5 min at 37oC. Cells were
treated with pronase and either separated into cytosolic and
membrane fractions, or cultured to assess viral growth and
neutralization. The fraction where nucleocapsid localized was
determined by antigen capture and microscopy techniques
were used to visualize HIV-1 within cells.
Results: TZMbl cells are 30-fold more efficient at internalizing
HIV-1 than PBMC with differences in entry observed between
viruses. In TZMbl cells, a greater fraction of p24 localized in the
membrane indicating endocytic entry, whereas in PBMC, SF162
localized predominantly in cytosol, indicating PM fusion, while Bal
localized equivalently in membrane and cytosolic fractions. Both
viruses are neutralized by 2G12 and b12, however, neutralization
by 2G12 was not associated with entry inhibition; in fact, after
the 5 minutes infection, entry appeared to be enhanced. In
contrast, b12 decreased viral entry.
Conclusion: Our data suggest that, for certain antibodies,
inhibition of HIV-1 may occur post-entry, and differences in
entry mechanisms may impact neutralization. Experiments
to elucidate the role of host restriction and post-entry
neutralization on inhibition of HIV-1 are ongoing.
124
AIDS Vaccine 2012
P03.16
Rate and Affinity Binding Constants Determined by
SPR Spectroscopy Reveal Differential Antigenicity of
HIV gp120 and gp140
T.L. Gearhart 1 , J.D. Steckbeck 1 , J.K. Craigo 1 , R.C. Montelaro 1
1 University of PIttsburgh, Pittsburgh, PA, USA
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 specificity 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 limitations of this format. To
provide a more sensitive, specific, and reproducible format
for our antibody assays, we have recently developed novel
procedures using SPR spectroscopy 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 identified significant distinguishing differences in several
binding characteristics of monoclonal antibody binding to gp120
vs gp140 antigens.
Conclusion: These kinetic parameters offer novel insights into the
fundamental interactions of reference monoclonal antibodies
and variant Env proteins and have the potential to provide novel
correlates of neutralization in vitro and protection in vivo.