Oral Abstract Session 01 - Global HIV Vaccine Enterprise

POSTERS
Posters
Topic 3: B Cell Immunology and Antibody Functions
P03.35
Antibody-Dependent Cellular Cytotoxicity-Mediating
Antibodies from an HIV-1 Vaccine Efficacy Trial
Preferentially Use the VH1 Gene Family
M. Bonsignori 1 , J. Pollara 1 , M.A. Moody 1 , T.B. Kepler 2 , X. Chen 1 ,
T.C. Gurley 1 , D.M. Kozink 1 , D.J. Marshall 1 , J.F. Whitesides 1 ,
J. Kaewkungwal 3 , S. Nitayaphan 4 , P. Pitisuttithum 5 , S. Rerks-
Ngarm 6 , J.H. Kim 7 , N.L. Michael 7 , D.C. Montefiori 1 , H. Liao 1 ,
G. Ferrari 1 , B.F. Haynes 1
1 Duke University Medical Center, Durham, NC, USA; 2 Boston
University School of Medicine, Boston, MA, USA; 3 Tropical
Hygiene, Mahidol University, Bangkok, Thailand; 4 Armed
Forces Research Institute of Medical Sciences, Bangkok,
Thailand; 5 Clinical Tropical Medicine, Mahidol University,
Bangkok, Thailand; 6 Department of Disease Control, Ministry
of Public Health, Nonthaburi, Thailand; 7 US Military HIV
Research Program, Rockville, MD, USA
Background: The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine efficacy
trial showed an estimated efficacy of 31%. The immune correlates
analysis raised the hypothesis that the observed protection in
RV144 may be partially due to Antibody-Dependent Cellular
Cytotoxicity (ADCC)-mediating antibodies in the presence of
low levels of Env IgA antibodies. In this study we analyzed the
Ig VH family usage of vaccine-induced ADCC mAbs isolated from
memory B cells of vaccinees.
Methods: From a total of 321,945 memory B-cells of 6 vaccinees
we obtained 23 mAbs that mediated ADCC using IgG+ memory
B-cell cultures (n=9) and Env-specific flow cytometric single
memory B-cell sorting (n=14). ADCC activity was measured using
both E.CM243 gp120-coated and E.CM235-infected target cells
in a flow-based assay.
Results: ADCC-mediating mAbs displayed a disproportionate
usage of VH1 family genes (17/23; 74%), in particular the VH1-
2 gene segment (10/17; 59%), as recently observed for CD4bs
broadly neutralizing antibodies (HAAD bNAbs). In contrast, only
17.1% of 111 heavy chains isolated from cultures that did not
mediate ADCC used the VH1 gene. VH1 ADCC-mediating mAbs
showed a high degree of V(D)J amino acid similarity to both the
VH (68-84%) and VL (70-87%) HAAD motifs. V(D)J rearrangements
displayed modest levels of affinity maturation (0.5-5.1% for
heavy chains and 0.4-4.3% for light chains). While none of the
VH1 ADCC-mediating mAbs was capable of mediating HIV-1
neutralization, the strength of their ADCC activity correlated
with the levels of heavy chain somatic mutations (p=0.02). We
produced the reverted unmutated ancestor antibodies of two
VH1 ADCC-mediating mAbs: one bound to B.MN Env and both
reacted against autoantigens.
Conclusion: ADCC-mediating antibodies induced by the ALVAC-
HIV/AIDSVAX-B/E vaccine underwent limited affinity maturation,
and preferentially used VH1 gene segments which share the
HAAD motif with CD4bs bNAbs. These observations raise the
hypothesis that HIV-1 Env preferentially selects VH1 family usage
for distinct subsets of antibodies with different functions.
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AIDS Vaccine 2012
P03.36
High-Resolution Crystal Structure of the Fv of
Quaternary Neutralizing Epitope mAb 2909 Reveals
Atomic Details of Its Antigen-Binding Site
J.M. Sampson 1 , A. Killikelly 1 , H. Zhang 1 , M.K. Gorny 1 , S. Zolla-
Pazner 1 , X. Kong 1
1 NYU School of Medicine, New York, NY, USA
Background: Human mAb 2909 is in a class of potently
neutralizing mAbs against the HIV-1 quaternary neutralizing
epitope (QNE) preferentially presented by the native Env trimer
complex. Its distinctive feature is a long CDR H3 loop with 2
sulfated tyrosines that are suggested to play a key role in antigen
binding. Two structures of the Fab fragment of 2909 have been
published, but at only 3.3Å and 3.2Å resolution, respectively,
some atomic-level details of the antigen binding sites of these
structures are contradictory.
Methods: After crystallizing a recombinant Fv (rFv) of mAb 2909,
expressed as a single chain in E. coli and refolded from inclusion
bodies, we solved and refined its structure to 1.9Å resolution.
We also characterized the neutralizing activity of the rFv against
pseudotyped virus SF162.
Results: Despite lacking the native sulfation of 2 tyrosine residues
at the apex of CDR H3, rFv 2909 retains neutralization activity
against SF162 pseudoviruses. Our high-resolution structure
features a series of 5 tyrosine residues decorating one face of H3
like rungs of a spiral staircase, as seen in the Spurrier structure.
The presence of this feature, despite different crystal packing
around the H3 loop, suggests that the stacking pattern is not an
artifact of crystallization, and that these tyrosine side chains play
an important role in epitope recognition.
Conclusion: Our structure of rFv 2909 at 1.9Å resolution reveals
additional atomic-level details of its antigen-binding site, allowing
further analysis of its binding mode. Our data demonstrate that
rFv can be used as a tool to obtain high-resolution structures
of antigen-binding regions, and may be useful for experiments
requiring molecular weights smaller than that of a full Fab
fragment, such as ITC and NMR spectroscopy.