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1.  An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1 
The Journal of Clinical Investigation  2014;124(4):1835-1843.
Broadly HIV-1–neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1–infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1–infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient’s plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.
doi:10.1172/JCI73441
PMCID: PMC3973118  PMID: 24614107
2.  Heavy Chain-Only IgG2b Llama Antibody Effects Near-Pan HIV-1 Neutralization by Recognizing a CD4-Induced Epitope That Includes Elements of Coreceptor- and CD4-Binding Sites 
Journal of Virology  2013;87(18):10173-10181.
The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.
doi:10.1128/JVI.01332-13
PMCID: PMC3753989  PMID: 23843638
3.  Breadth of cellular and humoral immune responses elicited in rhesus monkeys by multi-valent mosaic and consensus immunogens 
Virology  2012;428(2):121-127.
To create an HIV-1 vaccine that generates sufficient breadth of immune recognition to protect against the genetically diverse forms of the circulating virus, we have been exploring vaccines based on consensus and mosaic protein designs. Increasing the valency of a mosaic immunogen cocktail increases epitope coverage but with diminishing returns, as increasingly rare epitopes are incorporated into the mosaic proteins. In this study we compared the immunogenicity of 2-valent and 3-valent HIV-1 envelope mosaic immunogens in rhesus monkeys. Immunizations with the 3-valent mosaic immunogens resulted in a modest increase in the breadth of vaccine-elicited T lymphocyte responses compared to the 2-valent mosaic immunogens. However, the 3-valent mosaic immunogens elicited significantly higher neutralizing responses to Tier 1 viruses than the 2-valent mosaic immunogens. These findings underscore the potential utility of polyvalent mosaic immunogens for eliciting both cellular and humoral immune responses to HIV-1.
doi:10.1016/j.virol.2012.03.012
PMCID: PMC3582221  PMID: 22521913
HIV-1 vaccine; Mosaic immunogen; T cell
4.  Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing 
Science (New York, N.Y.)  2011;333(6049):1593-1602.
Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1–infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.
doi:10.1126/science.1207532
PMCID: PMC3516815  PMID: 21835983
5.  HIV-1 Neutralization Coverage Is Improved by Combining Monoclonal Antibodies That Target Independent Epitopes 
Journal of Virology  2012;86(6):3393-3397.
HIV-1 neutralizing monoclonal antibodies (MAbs) define key targets for vaccine development and are being considered for passive prevention of infection. We analyzed the interaction of MAbs to two independent epitopes on the viral envelope glycoprotein. Potently neutralizing MAbs to the CD4 binding site and V1V2 region displayed no in vitro cross-competition and displayed additive, though not synergistic, neutralization activity. Predicted neutralization coverage of a combination of two MAbs reached 97% on a 208-isolate panel.
doi:10.1128/JVI.06745-11
PMCID: PMC3302320  PMID: 22258252
6.  HIV Type 1 Env Precursor Cleavage State Affects Recognition by Both Neutralizing and Nonneutralizing gp41 Antibodies 
Abstract
HIV-1 is relatively resistant to antibody-mediated neutralization; however, rare antibodies to the exterior envelope glycoprotein, gp120, and the transmembrane glycoprotein, gp41, can neutralize a broad array of isolates. Two antibodies, 2F5 and 4E10, are directed against the gp41 membrane proximal external region (MPER); however, the kinetic neutralization signature of these antibodies remains unresolved. Previously, we reported that the fully cleaved, cell surface envelope glycoproteins (Env) derived from the primary isolate, JR-FL, are well recognized exclusively by gp120-directed neutralizing ligands and not by nonneutralizing gp120 antibodies. However, the gp120 nonneutralizing antibodies can recognize HIV spikes that are rendered fully cleavage defective by site-directed mutagenesis. Here, we extended such analysis to gp41 neutralizing and nonneutralizing antibodies and, relative to the rules of gp120-specific antibody recognition, we observed marked contrasts. Similar to gp120 recognition, the nonneutralizing gp41 cluster 1 or cluster 2 antibodies bound much more efficiently to cleavage-defective spikes when compared to their recognition of cleaved spikes. In contrast to gp120 neutralizing antibody recognition, the broadly neutralizing gp41 antibodies 2F5 and 4E10, like the nonneutralizing gp41 antibodies, did not efficiently recognize the predominantly cleaved, primary isolate JR-FL spikes. However, if the spikes were rendered cleavage defective, recognition by both the neutralizing and nonneutralizing ligand markedly increased. CD4 interaction with the cleaved spikes markedly increased recognition by most nonneutralizing gp41 antibodies, whereas such treatment had a minimal increase of 2F5 and 4E10 recognition. These data indicate again the profound influence that cleavage imposes on the quaternary packing of primary isolate spikes and have important implications for soluble trimer candidate immunogens.
doi:10.1089/aid.2010.0281
PMCID: PMC3159124  PMID: 21158699
7.  Mechanism of Neutralization by the Broadly Neutralizing HIV-1 Monoclonal Antibody VRC01▿† 
Journal of Virology  2011;85(17):8954-8967.
The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike.
doi:10.1128/JVI.00754-11
PMCID: PMC3165784  PMID: 21715490
8.  Rational Design of Envelope Identifies Broadly Neutralizing Human Monoclonal Antibodies to HIV-1 
Science (New York, N.Y.)  2010;329(5993):856-861.
Cross-reactive neutralizing antibodies (NAbs) are found in the sera of many HIV-1–infected individuals, but the virologic basis of their neutralization remains poorly understood. We used knowledge of HIV-1 envelope structure to develop antigenically resurfaced glycoproteins specific for the structurally conserved site of initial CD4 receptor binding. These probes were used to identify sera with NAbs to the CD4-binding site (CD4bs) and to isolate individual B cells from such an HIV-1–infected donor. By expressing immunoglobulin genes from individual cells, we identified three monoclonal antibodies, including a pair of somatic variants that neutralized over 90% of circulating HIV-1 isolates. Exceptionally broad HIV-1 neutralization can be achieved with individual antibodies targeted to the functionally conserved CD4bs of glycoprotein 120, an important insight for future HIV-1 vaccine design.
doi:10.1126/science.1187659
PMCID: PMC2965066  PMID: 20616233
9.  Relationship between Antibody 2F5 Neutralization of HIV-1 and Hydrophobicity of Its Heavy Chain Third Complementarity-Determining Region ▿ † ‡  
Journal of Virology  2009;84(6):2955-2962.
The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (P < 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 ∼10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.
doi:10.1128/JVI.02257-09
PMCID: PMC2826063  PMID: 20042512

Results 1-9 (9)