Fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gp120

Alanine scanning mutagenesis was performed on monomeric gp120 of human immunodeficiency virus type 1 to systematically identify residues important for gp120 recognition by neutralizing and nonneutralizing monoclonal antibodies (MAbs) to the CD4 binding site (CD4bs). Substitutions that affected the binding of broadly neutralizing antibody b12 were compared to substitutions that affected the binding of CD4 and of two nonneutralizing anti-CD4bs antibodies (b3 and b6) with affinities for monomeric gp120 comparable to that of b12. Not surprisingly, the sensitivities to a number of amino acid changes were similar for the MAbs and for CD4. However, in contrast to what was seen for the MAbs, no enhancing mutations were observed for CD4, suggesting that the virus has evolved toward an optimal gp120-CD4 interaction. Although the epitope maps of the MAbs overlapped, a number of key differences between b12 and the other two antibodies were observed. These differences may explain why b12, in contrast to nonneutralizing antibodies, is able to interact not only with monomeric gp120 but also with functional oligomeric gp120 at the virion surface. Neutralization assays performed with pseudovirions bearing envelopes from a selection of alanine mutants mostly showed a reasonable correlation between the effects of the mutations on b12 binding to monomeric gp120 and neutralization efficacy. However, some mutations produced an effect on b12 neutralization counter to that predicted from gp120 binding data. It appears that these mutations have different effects on the b12 epitope on monomeric gp120 and functional oligomeric gp120. To determine whether monomeric gp120 can be engineered to preferentially bind MAb b12, recombinant gp120s were generated containing combinations of alanine substitutions shown to uniquely enhance b12 binding. Whereas b12 binding was maintained or increased, binding by five nonneutralizing anti-CD4bs MAbs (b3, b6, F105, 15e, and F91) was reduced or completely abolished. These reengineered gp120s are prospective immunogens that may prove capable of eliciting broadly neutralizing antibodies.     

Design of a Non-glycosylated Outer Domain-derived HIV-1 gp120 Immunogen That Binds to CD4 and Induces Neutralizing Antibodies

The outer domain (OD) of the HIV-1 envelope glycoprotein gp120 is an important target for vaccine design as it contains a number of conserved epitopes, including a large fraction of the CD4 binding site. Attempts to design OD-based immunogens in the past have met with little success. We report the design and characterization of an Escherichia coli-expressed OD-based immunogen (OD_EC), based on the sequence of the HxBc2 strain. The OD_EC-designed immunogen lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gp120. Biophysical studies showed that OD_EC is folded and protease-resistant, whereas OD_EC lacking the designed mutations is highly aggregation-prone. In contrast to previously characterized OD constructs, OD_EC bound CD4 and the broadly neutralizing antibody b12 but not the non-neutralizing antibodies b6 and F105. Upon immunization in rabbits, OD_EC was highly immunogenic, and the sera showed measurable neutralization for four subtype B and one subtype C virus including two b12-resistant viruses. In contrast, sera from rabbits immunized with gp120 did not neutralize any of the viruses. OD_EC is the first example of a gp120 fragment-based immunogen that yields significant neutralizing antibodies.     

Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody.

While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gp120 epitopes recognized by neutralizing antibodies in infected individuals. Here, the epitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient was characterized by examining the effects of changes in conserved gp120 amino acids on antibody reactivity. The F105 epitope was disrupted by changes in gp120 amino acids 256 and 257, 368 to 370, 421, and 470 to 484, which is consistent with the discontinuous nature of the epitope. Three of these regions are proximal to those previously shown to be important for CD4 binding, which is consistent with the ability of the F105 antibody to block gp120-CD4 interaction. Since F105 recognition was more sensitive to amino acid changes in each of the four identified gp120 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gp120 epitope that is immunogenic in man and may serve as a target for therapeutic or prophylactic intervention.     

The C108g epitope in the V2 domain of gp120 functions as a potent neutralization target when introduced into envelope proteins derived from human immunodeficiency virus type 1 primary isolates

Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection.     

Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120-M9: implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4D12 and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4D12 to 17b is not affected by the addition of excess soluble CD4D12, while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4D12. Immunogenicity studies of gp120, gp120-CD4D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type 1 isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4D12. These antisera were depleted of anti-CD4D12 antibodies by being passed over a column containing immobilized CD4D12. The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4D12 competed with the CD4i antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4i epitope were generated by the gp120-CD4D12 immunogen, these antibodies were nonneutralizing.     

The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5

The human monoclonal antibody 2F5 neutralizes primary human immunodeficiency virus type 1 (HIV-1) with rare breadth and potency. A crystal structure of a complex of 2F5 and a peptide corresponding to its core epitope on gp41, ELDKWAS, revealed that the peptide interacts with residues at the base of the unusually long (22-residue) third complementarity-determining region of the heavy chain (CDR H3) but not the apex. Here, we perform alanine-scanning mutagenesis across CDR H3 and make additional substitutions of selected residues to map the paratope of Fab 2F5. Substitution of residues from the base of the H3 loop or from CDRs H1, H2, and L3, which are proximal to the peptide, significantly diminished the affinity of Fab 2F5 for gp41 and a short peptide containing the 2F5 core motif. However, nonconservative substitutions to a phenylalanine residue at the apex of the H3 loop also markedly decreased 2F5 binding to both gp41 and the peptide, suggesting that recognition of the core epitope is crucially dependent on features at the apex of the H3 loop. Furthermore, substitution at the apex of the H3 loop had an even more pronounced effect on the neutralizing activity of 2F5 against three sensitive HIV-1. These observations present a challenge to vaccine strategies based on peptide mimics of the linear epitope.     

Allocation of helper T-cell epitope immunodominance according to three-dimensional structure in the human immunodeficiency virus type I envelope glycoprotein gp120

The specificity and intensity of CD4(+) helper T-cell responses determine the effectiveness of immune effector functions. Promiscuously immunodominant helper T-cell epitopes in the human immunodeficiency virus (HIV) envelope glycoprotein gp120 could be important in the development of broadly protective immunity, but the underlying mechanisms of immunodominance and promiscuity remain poorly defined. In this study, gp120 helper T-cell epitopes were systematically mapped in CBA/J and BALB/c mice by restimulation assays using a set of overlapping peptides spanning the entire sequence of the gp120 encoded by HIV strain 89.6. The results were analyzed in the context of the HIV gp120 structure determined by x-ray crystallography. One major finding was that all of the promiscuously immunodominant gp120 sequences are located in the outer domain. Further analyses indicated that epitope immunogenicity in the outer domain correlates with structural disorder in adjacent N-terminal segments, as indicated by crystallographic B-factors or sequence divergence. In contrast, the correlation was poor when the analysis encompassed the entire gp120 sequence or was restricted to only the inner domain. These findings suggest that local disorder promotes the processing and presentation of adjacent epitopes in the outer domain of gp120 and therefore reveal how three-dimensional structure shapes the profile of helper T-cell epitope immunogenicity.     

Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.     

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

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.The human immunodeficiency virus type 1 (HIV-1) envelope is composed of surface gp120 and transmembrane gp41. Initial attempts to develop HIV vaccines through the induction of antibodies focused on recombinant gp120 glycoproteins. Two phase III clinical trials conducted in the United States and Thailand showed no protection from a gp120-based subunit vaccine against HIV infection, nor did the vaccine delay HIV-1 disease progression (11, 25). In addition, a phase II trial completed in Thailand with a live recombinant HIV-1 canarypox vaccine (vCP1452) in combination with a gp120 subunit protein did not stimulate a markedly improved immune response (28). The lack of efficacy was likely to be related to its failure to elicit broadly neutralizing antibodies (4, 10, 33).Several broadly neutralizing human monoclonal antibodies (MAbs) have been derived from infected individuals, including immunoglobulin G1 (IgG1) b12, 2G12, 2F5 and 4E10, which are directed against CD4-binding-site (BS), carbohydrate, and membrane-proximal regions of HIV Env (reviewed in reference 9). Among the most potent, the b12 antibody occludes the site of CD4 binding on gp120 and prevents virus attachment to CD4 on target cells (39). Other CD4-BS antibodies recognize epitopes on monomeric gp120 that overlap with b12 but lack the ability of b12 to neutralize primary HIV-1 isolates (5). An understanding of the specificity of b12 binding, neutralization, and protection should aid in the development of immunogens that induce neutralizing antibodies of a similar specificity.The structure of the b12-gp120 complex (39) shows that b12 binds to a conformationally conserved surface, which is centered around the CD4-binding loop on the outer domain of gp120. In the CD4-bound conformation of gp120, the CD4-binding loop or β15-strand makes antiparallel intermolecular hydrogen bonds to the C″ strand of CD4 (14). Overall, the outer domain of gp120 comprises 82% of the gp120 contact surface with b12, while most of the contacts outside of the outer domain have marginal importance (39). One exception, however, are contacts to the loop connecting the outer domain with the α5-helix of the inner domain (39), which appear to be important.Because it represents the smallest structural unit containing the b12 epitope, and therefore maximizes the b12-immunogenic surface relative to the overall surface, an outer domain-only immunogen with high b12 affinity represents an attractive immunogen. An outer domain construct (named OD1) was previously derived from HIV-1 strain YU2 gp120 and found to bind 2G12 and a number of anti-V3 antibodies (36); however, b12 binding to this construct was difficult to detect by enzyme-linked immunosorbent assay, probably due to an enhanced off rate (36, 39). A large, relatively flat interface exists between the inner and outer domains of gp120 in both CD4-bound and b12-bound conformations. We reasoned that the removal of the inner domain might partially destabilize it and decided to replace the inner domain with another polar surface, the cell membrane. We expressed outer domain proteins (ODs) in various membrane-anchored forms and tested their abilities to bind b12. An HIV-1 clade B R5 and X4 dual-tropic virus, R3A, was selected as a prototype (20). Laboratory-adapted virus strain R3A TA1 contains a truncated V1/V2 and a truncated V3 (named 9,9), maintains CCR5 tropism, and is highly sensitive to b12 neutralization (15, 23). We used available atomic-level structures to model an R3A gp120 core and to design truncations of flexible, potentially immunodominant structures, which emanate from OD, including the β20-β21 hairpin and the V3 loop. Thus, by using structure-based design to modify the OD form of R3A TA1, we attempted to remove strain-specific determinants, to enhance cell-surface expression, and to increase specific b12 binding compared to other native forms.     

Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120

IgG1 b12 is a broadly neutralizing antibody against human immunodeficiency virus type 1 (HIV-1). The epitope recognized by b12 overlaps the CD4 receptor-binding site (CD4bs) on gp120 and has been a target for vaccine design. Determination of the three-dimensional structure of immunoglobulin G1 (IgG1) b12 allowed modeling of the b12-gp120 interaction in which the protruding third complementarity-determining region (CDR) of the heavy chain (H3) was crucial for antibody binding. In the present study, extensive mutational analysis of the antigen-binding site of Fab b12 was carried out to investigate the validity of the model and to identify residues important for gp120 recognition and, by inference, key to the anti-HIV-1 activity of IgG1 b12. In all, 50 mutations were tested: 40 in H3, 4 each in H2 and L1, and 2 in L3. The results suggest that the interaction of gp120 with H3 of b12 is crucially dependent not only on a Trp residue at the apex of the H3 loop but also on a number of residues at the base of the loop. The arrangement of these residues, including aromatic side chains and side chains that hydrogen bond across the base of the loop, may rigidify H3 for penetration of the recessed CD4-binding cavity. The results further emphasize the importance to gp120 binding of a Tyr residue at the apex of the H2 loop that forms a second finger-like structure and a number of Arg residues in L1 that form a positively charged, shelf-like structure. In general, the data are consistent with the b12-gp120 interaction model previously proposed. At the gene level, somatic mutation is seen to be crucial for the generation of many of the structural features described. The Fab b12 mutants were also tested against the b12 epitope-mimic peptide B2.1, and the reactivity profile had many similarities but also significant differences from that observed for gp120. The paratope map of b12 may facilitate the design of molecules that are able to elicit b12-like activities.     

Crystal structure of a 3B3 variant—A broadly neutralizing HIV‐1 scFv antibody

We present the crystal structure determination of an anti‐HIV‐1 gp120 single‐chain variable fragment antibody variant, 3B3, at 2.5 Å resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site‐directed mutagenesis of the variable heavy chain (V_H) complementary‐determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross‐clade primary isolates of HIV‐1 by interaction with the recessed CD4‐binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR‐H3 of the V_H domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR‐H3 of 3B3, in light of the b12‐gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR‐L3 of the variable light (V_L) domain triggered by a point mutation in CDR‐H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the V_L and V_H domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3‐gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the V_L and V_H domains possibly through more favorable entropic effect through the expulsion of water.     

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C' and FG. This structure is consistent with C'C' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.     

Dramatic Potentiation of the Antiviral Activity of HIV Antibodies by Cholesterol Conjugation

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10–100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.     

Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1

The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.     

Designed cyclic permutants of HIV-1 gp120: implications for envelope trimer structure and immunogen design

Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120-gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. We have therefore designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with ～20-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface.     

Hyperglycosylated mutants of human immunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIV vaccine design

The ability to induce broadly neutralizing antibodies should be a key component of any forthcoming vaccine against human immunodeficiency virus type 1. One potential vaccine candidate, monomeric gp120, has generally failed to elicit such antibodies. We postulated that gp120 might be a better immunogen if it could be engineered to preferentially bind known broadly neutralizing antibodies. In a first study, we found that four alanine substitutions on the perimeter of the so-called Phe-43 cavity of gp120 could reduce binding of weakly neutralizing CD4-binding site antibodies (R. Pantophlet, E. O. Saphire, P. Poignard, P. W. H. I. Parren, I. A. Wilson, and D. R. Burton, J. Virol. 77:642-658, 2003), while slightly enhancing binding of the potent, broadly neutralizing antibody b12. In the present study, we sought to reduce or abolish the binding of a wider range of nonneutralizing antibodies, by incorporating extra N-glycosylation motifs at select positions into the hypervariable loops and the gp120 core. A hyperglycosylated mutant containing seven extra glycosylation sequons (consensus sequences) and the four alanine substitutions described above did not bind an extensive panel of nonneutralizing and weakly neutralizing antibodies, including a polyclonal immunoglobulin preparation (HIVIG) of low neutralizing potency. Binding of b12, at lowered affinity, and of four antibodies to the C1 and C5 regions was maintained. Removal of N- and C-terminal residues in the C1 and C5 regions, respectively, reduced or abolished binding of the four antibodies, but this also adversely affected b12 binding. The hyperglycosylated mutant and its analogues described here are novel antigens that may provide a new approach to eliciting antibodies with b12-like neutralizing properties.     

Antigenic Properties of the HIV Envelope on Virions in Solution

The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro.     

Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.     

Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently.     

Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope

The human monoclonal antibody Fab X5 neutralizes a broad range of HIV-1 primary isolates. The crystal structure of X5 has been determined at 1.9 A resolution. There are two crystallographically independent Fab fragments in the asymmetric unit. The crystallographic R value for the final model is 0.22. The antibody-combining site features a long (22 amino acid residues) CDR H3 with a protruding hook-shaped motif. The X5 structure and site-directed mutagenesis data suggest that X5 amino acid residues W100 and Y100F in the CDR H3 motif may be critical for the binding of Fab X5 to gp120. X5 bound to a complex of a CD4 mimetic and gp120 with approximately the same kinetics and affinity as to a CD4-gp120 complex, suggesting that specific interactions between CD4 and X5 are unlikely to contribute to the binding of X5 to gp120-CD4 complexes. Binding of X5 to alanine scanning mutants of gp120JR-CSF complexed with CD4 suggested a critical role of the highly conserved amino acid residues at positions 423 and 432. The X5 structure and fine mapping of its epitope may assist in the elucidation of the mechanisms of viral entry and neutralization, and the development of HIV-1 inhibitors and vaccines.