Oral Abstract Session 01 - Global HIV Vaccine Enterprise

Topic 3: B Cell Immunology and Antibody Functions
P03.45
Conformational Study of Quaternary Epitope Region
of V1/V2 Loop: Influence of Disulfide Bonds and
Glycosylations
G. Gnanakaran 1 , J. Tian 1 , A. Sethi 1
1 Los Alamos National Labs, Los Alamos, USA
Background: The HIV-1 envelope spike, which consists of a
compact, heterodimeric trimer of the glycoprotein gp120 and
gp41, is the sole viral target of neutralizing antibodies. The gp120
component of the viral spike is known to be heavily glycosylated,
and glycosylation can affect the conformation of envelope spikes.
V1/V2 variable loops of gp120 are key target regions for a number
of broadly neutralizing human antibodies, such as PG9 and PG16,
CH01-CH04, and PGT141-145. Two glycosylation sites (N156 and
N160) have been shown by mutagenesis studies to be important
in forming the PG9 and PG16 epitopes. Recently, Peter Kwong
and coworkers have resolved crystal structure of V1/V2 domain
of HIV-1 gp120 from strains CAP45 and ZM109 complexes with
antigen-binding fragment of PG9.
Methods: We employ enhanced molecular dynamics sampling
methods (eg. replica exchange molecular dynamics) to dissect
the influence of disulfide bond and glycosylation on the
conformational landscape of an indel free epitope region of V1/
V2 loop. These methods are expected to capture the influence
of glycosylation, solvent, rest of the gp120 protein and scaffold
constructs on the conformation of V1/V2.
Results: We evaluate the backbone conformational preferences
and solvent accessibility of each residue in the selected V1/V2
region and compare them to the antibody-bound conformation
of this region. Both the disulfide bond that links V1 and V2 loops
and the nearby glycosylations affect the beta sheet formation
propensity of that region. Further characterization indicates that
glycans predominantly influence the entropy of the V1/V2 loops.
Conclusion: Our studies reveal how glycans can impact the
electrostatic and hydrophobic surfaces of the V1/V2 regions that
have been proposed to form the epitope for broadly neutralizing
antibodies. Along with proximal disulfide bonds, glycans tend
to affect the local beta-sheet propensities that can further
contribute to the quaternary nature of the epitope.
P03.46
AIDS Vaccine 2012
Posters
Short Constrained Peptides Derived from Phage
Display Libraries as Epitope Models: The Case of mAb
2F5
Y. Palacios-Rodriguez 1 , T. Gazarian 2 , L. Huerta 2 , K. Gazarian 2
1 Mexican National Autonomous University, Mexico,
D.F., Mexico; 2 Institute of Biomedical Research UNAM,
Mexico DF, Mexico
Background: Since the monoclonal antibody 2F5 (mAb 2F5) was
isolated in the early 90’s, its epitope have continued to be the
focus of extensive investigations attempting to elucidate the
mechanism by which impedes viral entry into host cells. Because
the DKW-flanking amino acids are strongly conserved in viruses,
it is not clear whether the DKW only satisfies the 2F5 epitope
recognition demand.
Methods: We used phage display technology involving
biopanning of a pIII-type 7-mer constrained peptide library
(not screened in previous experiments with 2F5) for its epitope
mimics. After peptides selection and widely characterization
of several phage-peptide clones, some of them were used as
immunogens. Polyclonal antibodies were evaluated as cell-cell
fusion inhibitors of the CD4-Env complex interactions.
Results: We found that the specificity of recognition of the
epitope depends on the structural context in which the cognate
epitope sequence is presented. The antibody does not tolerate
any replacements of the DKW-flanking epitope amino acids and
binds exclusively to the (L)DKWA sequence provided by a 7-mer
constrained peptide exposed by the M13 phage pIII protein.
Additionally, immunization data supports the notion that the
binding and neutralizing immunogenic structural features of the
described epitope model do not coincide.
Conclusion: In this study, we show that when mAb 2F5 screens
a pIII-type phage display 7-mer constrained peptide library for
its epitope mimics, it demands an epitope sequence longer than
DKW and does not tolerate substitutions in the epitope amino
acid sequence as has been suggested in previous reports.
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POSTERS