Oral Abstract Session 01 - Global HIV Vaccine Enterprise

POSTERS
Posters
Topic 3: B Cell Immunology and Antibody Functions
P03.07
Design of Epitope-Specific Probes for Sera Analysis
and Antibody Isolation
I. Georgiev 1 , P. Acharya 1 , S.D. Schmidt 1 , Y. Li 2 , D. Wycuff 3 ,
G. Ofek 1 , N. Doria-Rose 1 , T.S. Luongo 1 , Y. Yang 1 , T. Zhou 1 ,
B.R. Donald 4 , J.R. Mascola 1 , P.D. Kwong 1
1 Vaccine Research Center/NIAID/NIH, Bethesda, MD, USA;
2 The Scripps Research Institute, La Jolla, CA, USA; 3 National
Institutes of Health, Bethesda, MD, USA; 4 Duke University,
Durham, NC, USA
Background: The design of gp120 monomeric probes with
modified antigenic profiles that are specific for a target epitope
has been successfully used for the isolation of broadly neutralizing
HIV-1 antibodies. Existing probes, however, do not possess
sufficient specificity and can bind antibodies with undesired
properties (e.g., weakly neutralizing antibodies targeting an
overlapping epitope). To achieve improved epitope specificity,
positive and negative design stages can be incorporated into the
probe design process.
Methods: Here, we apply a combination of structure- and
sequence-based methods for improving the epitope specificity
of gp120 monomeric probes. Specifically, structure-based
redesign using the OSPREY protein design software suite
was used to predict gp120 knock-out mutations for selected
antibodies. Additionally, using a sequence-based mutual
information approach, knock-out mutations were designed by
identifying gp120 residues that are predicted to associate with
neutralization resistance for a given antibody.
Results: Using stabilized (Ds12F123) and resurfaced stabilized
(RSC3) HXB2 gp120 cores as templates, we designed a set of
mutants with improved epitope specificity. In particular, RSC3 (a
prototypic probe previously used for the isolation of VRC01 and
other CD4-binding-site antibodies) was redesigned to specifically
bind CD4-binding-site antibodies that are broadly neutralizing
(VRC01, VRC-PG04: positive design) but not moderately/weakly
neutralizing (b12, b13, HJ16: negative design). Additionally,
CD4i-specific probes were designed by introducing mutations
that destabilize binding to the entire class of CD4-binding-site
antibodies (negative design), while retaining binding to CD4i
antibodies (positive design). The desired epitope specificity of
the redesigned probes was confirmed by ELISA binding. The
probe design approach was further validated with knock-out
mutations for a diverse set of antibodies, including PG9 and 2F5.
Conclusion: Probes with enhanced epitope specificity can select
more precisely for antibodies with desired properties. A set
of our redesigned probes are currently being utilized for the
isolation of antibodies targeting different epitopes of interest on
the HIV-1 Envelope.
120
AIDS Vaccine 2012
P03.08
Critical Role for Monocytes in Mediating HIV-Specific
Antibody-Dependent Cellular Cytotoxicity
M. Kramski 1 , G.F. Lichtfuss 2 , A. Schorcht 1 , A.P. Johnston 1 ,
R. De Rose 1 , R. Center 1 , A. Jawarowski 2 , S. Kent 1
1 University of Melbourne, Melbourne, Australia; 2 Burnet
Institute, Melbourne, Australia
Background: Antibodies (Abs) that mediate antibody-dependent
cellular cytotoxicity (ADCC) activity against HIV-1 are of major
interest. Considerable evidence supports a role for ADCC activity
in the control of HIV-1 infection and in the context of vaccination.
One method widely used to assess the role of ADCC is the rapid
and fluorometric antibody-dependent cellular cytotoxicity
(RFADCC) assay. In the RFADCC assay specific killing of target cells
by PBMC is assessed by loss of intracellular CFSE but retention
of membrane dye PKH26 (CFSE-PKH26+), which is assumed to
be derived from CFSE+PKH26+ target cells killed by NK cells. We
have revisited this assay to assess the role of effector cells in
mediating ADCC.
Methods: Multi-color flow cytometry was used to analyse gp140pulsed,
CFSE and PKH26 double labeled CEM.NKr-CCR5 cells
incubated with HIV+ plasma or purified IgG samples (n=57) and
co-cultured with PBMC, purified NK cells, or monocytes prepared
from healthy donor blood. Effector/target cell interaction was
visualized using image stream flow cytometry and live cell imaging.
Results: Backgating analysis and phenotyping of CFSE-PKH26+
cells identified CD3-CD14+ monocytes as the major effector cell
type. This was confirmed for all 57 HIV+ plasma samples tested.
Emergence of the CFSE-PKH26+ cell population was observed
following co-culture with purified monocytes but not purified
NK cells. No significant IFNγ production or CD107a degranulation
was detected in NK cells in this assay. Image flow cytometry
and microscopy confirmed a monocyte-specific interaction
with target cells. Monocytes acquire PKH26+ cell membrane
presumably derived from killed target cells without typical
morphological changes associated with phagocytosis, suggesting
monocyte-mediated ADCC.
Conclusion: Our studies advance the understanding of the
cellular events underlying HIV-specific ADCC. The RFADCC assay
primarily reflects Ab-mediated monocyte function and has to be
treated with caution in regard to NK cell-mediated ADCC. Further
studies on the biological importance of HIV-specific monocytemediated
ADCC are warranted.