Efficiency of Neutralizing Antibodies Targeting the CD4-Binding Site: Influence of Conformational Masking by the V2 Loop in R5-Tropic Clade C Simian-Human Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) gp120 exterior glycoprotein is conformationally flexible. Upon binding the host cell receptor, CD4, gp120 assumes a conformation that is able to bind the chemokine receptors CCR5 or CXCR4, which act as coreceptors for the virus. CD4-binding-site (CD4BS) antibodies are neutralizing antibodies elicited during natural infection that are directed against gp120 epitopes that overlap the binding site for CD4. Recent studies (S. H. Xiang et al., J. Virol. 76:9888-9899, 2002) suggest that CD4BS antibodies recognize conformations of gp120 distinct from the CD4-bound conformation. This predicts that the binding of CD4BS antibodies will inhibit chemokine receptor binding. Here, we show that Fab fragments and complete immunoglobulin molecules of CD4BS antibodies inhibit CD4-independent gp120 binding to CCR5 and cell-cell fusion mediated by CD4-independent HIV-1 envelope glycoproteins. These results are consistent with a model in which the binding of CD4BS antibodies limits the ability of gp120 to assume a conformation required for coreceptor binding.

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Comprehensive Cross-Clade Neutralization Analysis of a Panel of Anti-Human Immunodeficiency Virus Type 1 Monoclonal Antibodies

Journal of Virology ◽

10.1128/jvi.78.23.13232-13252.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13232-13252 ◽

Cited By ~ 553

Author(s):

James M. Binley ◽

Terri Wrin ◽

Bette Korber ◽

Michael B. Zwick ◽

Meng Wang ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Monoclonal Antibodies ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Clade B ◽

Immunodeficiency Virus ◽

Clade C ◽

Cd4 Binding Site

ABSTRACT Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV+ plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (≤7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV+ plasma neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutralizing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design, our data have implications for passive-immunization studies in countries where clade C viruses are common, given that only MAbs b12 and 4E10 were effective against viruses from this clade.

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Enhanced Exposure of the CD4-Binding Site to Neutralizing Antibodies by Structural Design of a Membrane-Anchored Human Immunodeficiency Virus Type 1 gp120 Domain

Journal of Virology ◽

10.1128/jvi.02600-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 5077-5086 ◽

Cited By ~ 40

Author(s):

Lan Wu ◽

Tongqing Zhou ◽

Zhi-yong Yang ◽

Krisha Svehla ◽

Sijy O'Dell ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Neutralizing Antibodies ◽

Outer Domain ◽

Immunodeficiency Virus ◽

Cd4 Binding Site ◽

Hiv 1

ABSTRACT The broadly neutralizing antibody immunoglobulin G1 (IgG1) b12 binds to a conformationally conserved surface on the outer domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) glycoprotein. To develop outer domain proteins (ODs) that could be recognized selectively by CD4-binding-site (CD4-BS) antibodies, membrane-anchored ODs were generated from an HIV-1 clade B virus, TA1 R3A, which was highly sensitive to neutralization by the IgG1 b12 antibody. A 231-residue fragment of gp120 (residues 252 to 482) linked to transmembrane regions from CD4 showed b12 binding comparable to that of the native Env spike as measured by flow cytometry. Truncation of the β20-β21 hairpin (residues 422 to 436 to Gly-Gly) improved overall protein expression. Replacement of the immunodominant central 20 amino acids of the V3 loop (residues 302 to 323) with a basic hexapeptide (NTRGRR) increased b12 reactivity further. Surface calculations indicated that the ratio of b12 epitope to exposed immunogenic surface in the optimized OD increased to over 30%. This OD variant [OD(GSL)(Δβ20-21)(hCD4-TM)] was recognized by b12 and another CD4-BS-reactive antibody, b13, but not by eight other CD4-BS antibodies with limited neutralization potency. Furthermore, optimized membrane-anchored OD selectively absorbed neutralizing activity from complex antisera and b12. Structurally designed membrane-anchored ODs represent candidate immunogens to elicit or to allow the detection of broadly neutralizing antibodies to the conserved site of CD4 binding on HIV-1 gp120.

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Factors Associated with the Development of Cross-Reactive Neutralizing Antibodies during Human Immunodeficiency Virus Type 1 Infection

Journal of Virology ◽

10.1128/jvi.02036-08 ◽

2008 ◽

Vol 83 (2) ◽

pp. 757-769 ◽

Cited By ~ 378

Author(s):

D. Noah Sather ◽

Jakob Armann ◽

Lance K. Ching ◽

Angeliki Mavrantoni ◽

George Sellhorn ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Variable Regions ◽

Factors Associated ◽

Immunodeficiency Virus ◽

Cd4 Binding Site

ABSTRACT The characterization of the cross-reactive, or heterologous, neutralizing antibody responses developed during human immunodeficiency virus type 1 (HIV-1) infection and the identification of factors associated with their generation are relevant to the development of an HIV vaccine. We report that in healthy HIV-positive, antiretroviral-naïve subjects, the breadth of plasma heterologous neutralizing antibody responses correlates with the time since infection, plasma viremia levels, and the binding avidity of anti-Env antibodies. Anti-CD4-binding site antibodies are responsible for the exceptionally broad cross-neutralizing antibody responses recorded only in rare plasma samples. However, in most cases examined, antibodies to the variable regions and to the CD4-binding site of Env modestly contributed in defining the overall breadth of these responses. Plasmas with broad cross-neutralizing antibody responses were identified that targeted the gp120 subunit, but their precise epitopes mapped outside the variable regions and the CD4-binding site. Finally, although several plasmas were identified with cross-neutralizing antibody responses that were not directed against gp120, only one plasma with a moderate breadth of heterologous neutralizing antibody responses contained cross-reactive neutralizing antibodies against the 4E10 epitope, which is within the gp41 transmembrane subunit. Overall, our study indicates that more than one pathway leads to the development of broad cross-reactive neutralizing antibodies during HIV infection and that the virus continuously escapes their action.

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Determinants of Neutralization Resistance in the Envelope Glycoproteins of a Simian-Human Immunodeficiency Virus Passaged In Vivo

Journal of Virology ◽

10.1128/jvi.73.10.8873-8879.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8873-8879 ◽

Cited By ~ 55

Author(s):

Bijan Etemad-Moghadam ◽

Ying Sun ◽

Emma K. Nicholson ◽

Gunilla B. Karlsson ◽

Dominik Schenten ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Neutralizing Antibodies ◽

Envelope Glycoproteins ◽

V3 Loop ◽

Variable Loop ◽

Immunodeficiency Virus ◽

Cd4 Binding Site ◽

Hiv 1

ABSTRACT In vivo passage of a simian-human immunodeficiency virus (SHIV-89.6) generated a virus, SHIV-89.6P, that exhibited increased resistance to some neutralizing antibodies (G. B. Karlsson et al., J. Exp. Med. 188:1159–1171, 1998). Here we examine the range of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies to which the passaged virus became resistant and identify envelope glycoprotein determinants of antibody resistance. Compared with the envelope glycoproteins derived from the parental SHIV-89.6, the envelope glycoproteins of the passaged virus were resistant to antibodies directed against the gp120 V3 variable loop and the CD4 binding site. By contrast, both viral envelope glycoproteins were equally sensitive to neutralization by two antibodies, 2G12 and 2F5, that recognize poorly immunogenic structures on gp120 and gp41, respectively. Changes in the V2 and V3 variable loops of gp120 were necessary and sufficient for full resistance to the IgG1b12 antibody, which is directed against the CD4 binding site. Changes in the V3 loop specified complete resistance to a V3 loop-directed antibody, while changes in the V1/V2 loops conferred partial resistance to this antibody. The epitopes of the neutralizing antibodies were not disrupted by the resistance-associated changes. These results indicate that in vivo selection occurs for HIV-1 envelope glycoproteins with variable loop conformations that restrict the access of antibodies to immunogenic neutralization epitopes.

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Characterization of Human Immunodeficiency Virus Type 1 Monomeric and Trimeric gp120 Glycoproteins Stabilized in the CD4-Bound State: Antigenicity, Biophysics, and Immunogenicity

Journal of Virology ◽

10.1128/jvi.02500-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5579-5593 ◽

Cited By ~ 80

Author(s):

Barna Dey ◽

Marie Pancera ◽

Krisha Svehla ◽

Yuuei Shu ◽

Shi-Hua Xiang ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Bound State ◽

Wild Type ◽

Gp120 Envelope ◽

Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S+S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S+S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.

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Structure of the Fab Fragment of F105, a Broadly Reactive Anti-Human Immunodeficiency Virus (HIV) Antibody That Recognizes the CD4 Binding Site of HIV Type 1 gp120

Journal of Virology ◽

10.1128/jvi.79.20.13060-13069.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 13060-13069 ◽

Cited By ~ 25

Author(s):

Royce A. Wilkinson ◽

Chayne Piscitelli ◽

Martin Teintze ◽

Lisa A. Cavacini ◽

Marshall R. Posner ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Neutralizing Antibody ◽

Binding Pocket ◽

Human Antibody ◽

Fab Fragment ◽

Outer Domain ◽

Immunodeficiency Virus ◽

Cd4 Binding Site ◽

The Difference

ABSTRACT We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe43 of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.

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V2 Loop Glycosylation of the Human Immunodeficiency Virus Type 1 SF162 Envelope Facilitates Interaction of This Protein with CD4 and CCR5 Receptors and Protects the Virus from Neutralization by Anti-V3 Loop and Anti-CD4 Binding Site Antibodies

Journal of Virology ◽

10.1128/jvi.74.15.6769-6776.2000 ◽

2000 ◽

Vol 74 (15) ◽

pp. 6769-6776 ◽

Cited By ~ 119

Author(s):

Amy Ly ◽

Leonidas Stamatatos

Keyword(s):

Human Immunodeficiency Virus ◽

Viral Replication ◽

Binding Site ◽

Target Cells ◽

V3 Loop ◽

Dependent Manner ◽

Immunodeficiency Virus ◽

Cd4 Binding Site ◽

Carboxy Terminal ◽

In Cells

ABSTRACT We examined the role of asparagine-linked glycosylation of the V2 loop of the human immunodeficiency virus (HIV) SF162 envelope on viral replication potential and neutralization susceptibility. We report that the asparagines located at the amino- and carboxy-terminal sites (at positions 154 and 195, respectively), as well as within the V2 loop of the SF162 envelope (at position 186), are glycosylated during in vitro replication of this virus in human peripheral blood mononuclear cells. Our studies indicate that glycosylation of the V2 loop, in particular at its base, facilitates the interaction of the HIV envelope with the CD4 and CCR5 receptor molecules present on the surface of target cells and affects viral replication kinetics in a cell type-dependent manner. In cells expressing high numbers of receptor molecules on their surfaces, the SF162-derived V2 loop-deglycosylated mutant viruses replicate as efficiently as the parental SF162 virus, while in cells expressing small numbers of receptor molecules, the mutant viruses replicate with markedly reduced efficiency. In addition to expanding the viral tropism, V2 loop glycosylation at the three sites examined prevents neutralization by anti-CD4 binding site antibodies. In contrast, glycosylation at the amino- and carboxy-terminal sites of the V2 loop but not within the loop itself offers protection against anti-V3 loop antibodies. Thus, the epitopes masked by the sugar molecules present on the three glycosylation sites examined are not identical but overlap.

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Changes in the Immunogenic Properties of Soluble gp140 Human Immunodeficiency Virus Envelope Constructs upon Partial Deletion of the Second Hypervariable Region

Journal of Virology ◽

10.1128/jvi.77.4.2310-2320.2003 ◽

2003 ◽

Vol 77 (4) ◽

pp. 2310-2320 ◽

Cited By ~ 92

Author(s):

Indresh K. Srivastava ◽

Keating VanDorsten ◽

Lucia Vojtech ◽

Susan W. Barnett ◽

Leonidas Stamatatos

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Hypervariable Region ◽

V3 Loop ◽

Virus Envelope ◽

Immunodeficiency Virus ◽

Cd4 Binding Site ◽

Hiv Envelope ◽

Immunogenic Properties ◽

Hiv 1

ABSTRACT Immunization of macaques with the soluble oligomeric gp140 form of the SF162 envelope (SF162gp140) or with an SF162gp140-derived construct lacking the central region of the V2 loop (ΔV2gp140) results in the generation of high titers of antibodies capable of neutralizing the homologous human immunodeficiency virus type 1 (HIV-1), SF162 virus (Barnett et al. J. Virol. 75 :5526-5540, 2001). However, the ΔV2gp140 immunogen is more effective than the SF162gp140 immunogen in eliciting the generation of antibodies capable of neutralizing heterologous HIV-1 isolates. This indicates that deletion of the V2 loop alters the immunogenicity of the SF162gp140 protein. The present studies were aimed at identifying the envelope regions whose immunogenicity is altered following V2 loop deletion. We report that the antibodies elicited by the SF162gp140 immunogen recognize elements of the V1, V2, and V3 loops, the CD4-binding site, and the C1 and C2 regions on the homologous SF162 gp120. With the exception of the V1 and V2 loops, the same regions are recognized on heterologous gp120 proteins. Surprisingly, although a minority of the SF162gp140-elicited antibodies target the V3 loop on the homologous gp120, the majority of the antibodies elicited by this immunogen that are capable of binding to the heterologous gp120s tested recognize their V3 loops. Deletion of the V2 loop has two effects. First, it alters the immunogenicity of the V3 and V1 loops, and second, it renders the C5 region immunogenic. Although deletion of the V2 loop does not result in an increase in the immunogenicity of the CD4-binding site per se, the relative ratio of anti-CD4-binding site to anti-V3 loop antibodies that bind to the heterologous gp120s tested is higher in sera collected from the ΔV2gp140-immunized animals than in the SF162gp140-immunized animals. Overall, our studies indicate that it is possible to alter the immunogenic structure of the HIV envelope by introducing specific modifications.

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