We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421–433 and 288–306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (V
H) domain framework (FR) residues. Substitution of the FR cavity V
H Lys-19 residue by an Ala residue resulted in attenuated binding of the 421–433 region peptide probe. The CDRs and V
H FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from V
H1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421–433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.Induction of neutralizing antibodies (Abs)
2 via adaptive immune processes is the cornerstone of vaccination against microbial antigens. The antigen-binding site is mostly formed by the complementarity determining regions (CDRs) of the light and heavy chain variable domains (V
L and V
H domains). Vaccine-induced adaptive Ab responses entail sequence diversification of Ab V domains expressed within the B cell receptor (BCR) complex, selective noncovalent antigen binding to the high affinity BCR mutants, and proliferation of the mutant B cell clones. No HIV vaccine is available. The surface of HIV is studded with noncovalently associated oligomers of gp120 complexed to gp41. HIV infection and experimental HIV vaccination attempts induce robust Ab responses to the immunodominant epitopes of gp120, which are structurally divergent in various HIV strains responsible for infection in different parts of the world. Abs to such epitopes express strain-specific neutralization (
1,
2),
i.e. they neutralize the HIV strain from which the immunogen was isolated but not strains genetically heterologous to the immunogen.The gp120 site responsible for binding host CD4 receptors (CD4BS) is structurally more conserved. Precise conformational details of the CD4BS expressed on the HIV surface are not available, but crystallography suggests a large, discontinuous determinant composed of regions distant from each other in the linear protein sequence (
3,
4). The 421–433 peptide region is essential for CD4 binding by gp120, suggested by contacts in the crystallized complex and loss of CD4 binding function by site-directed mutagenesis in this region (
5,
6). The 421–433 region is a member of a small group of microbial polypeptide sites recognized selectively by Abs produced by the immune system without prior infection by the microbe (preimmune Abs) (
7–
9). Such sites are designated B cell “superantigens” (SAgs) because of their selective and widespread recognition by the comparatively conserved framework regions (FRs) of Ab V domains (
10,
11). Noncovalent SAg binding by preimmune Abs, however, is characterized by low-to-moderate binding strength (
12). Most gp120-binding preimmune Abs from humans without infection display poor or no HIV neutralizing activity (
13). Patients with the autoimmune disease lupus and no HIV infection produce increased amounts of Abs to the 421–433 CD4BS region (
14). A single chain Fv (scFv; V
L and V
H domains linked by a flexible peptide) from the lupus Ab repertoire that binds the 421–433 region reversibly neutralizes genetically diverse strains of HIV (
15). Following completion of the noncovalent binding step, certain Abs can hydrolyze polypeptides via nucleophilic attack on carbonyl groups (
16–
21). The proteolytic reaction imparts improved antigen inactivation potency to Abs (
22). We reported the neutralization of HIV by secretory IgA from humans without infection, an Ab class distinguished by the ability to catalyze the hydrolysis of gp120 selectively because of initial noncovalent recognition of the 421–433 CD4BS region (
13).The conserved character of the CD4BS in genetically diverse HIV strains renders it suitable as a vaccine target. The CD4BS, however, is poorly immunogenic. Traditional immunization methods do not stimulate the adaptive synthesis of neutralizing Abs to the 421–433 region or other CD4BS epitopes. Neutralizing Abs that bind the CD4BS are found in the blood of a subset of patients after years of HIV infection, but the target epitope is not identified, and Ab response is weak (
23,
24). Certain monoclonal Abs (mAbs) that bind the CD4BS expressed by purified monomer gp120 do not neutralize HIV appreciably or display limited ability to neutralize genetically diverse HIV strains (
25,
26). The CD4BS is a flexible structure expressed in differing conformational states by monomer gp120 and the native gp120 oligomers of the virus (
27–
30). Moreover, the process of binding CD4 may induce movements within the CD4BS (
31). Reproducing the native CD4BS conformation in experimental vaccine candidates has been difficult. A CD4BS mimetic of the epitope recognized by a well known anti-CD4BS neutralizing mAb (clone b12) did not induce the synthesis of neutralizing Abs (
32). Polyclonal Abs raised by immunization with synthetic peptides spanning the 421–433 CD4BS region neutralized laboratory-adapted, coreceptor CXCR4-dependent HIV strains inconsistently (
33–
35). Neutralization of coreceptor CCR5-dependent strains responsible for initiating most HIV infections was not studied. Importantly, small synthetic peptides are often more flexible than the corresponding native protein segments. Inducing a traditional adaptive immune response in which the Ab CDRs develop binding specificity for the peptide immunogen therefore does not ensure recognition of the native 421–433 CD4BS region (
35,
36). From mutagenesis and sequence identity studies, the gp120-binding site of preimmune Abs, in contrast, is composed mainly of the V
H domain FR1 and FR3 (
10,
11,
37). As certain preimmune Abs express HIV neutralizing activity attributable to recognition of the 421–433 region (
13), the FR-dominated site must recognize the native state of this CD4BS epitope expressed on the viral surface.There is, however, substantial difficulty in amplifying and improving the subset of preimmune Abs with HIV neutralizing activity for vaccination against the virus; SAg binding to Ab FRs fails to stimulate adaptive B cell differentiation and synthesis of specific IgG class Abs (
38,
39). Indeed, the binding at the FRs may even lead to premature death of the B cells (
12,
40). The SAg character of the 421–433 CD4BS epitope is therefore predicted to render it hypoimmunogenic with respect to the adaptive synthesis of neutralizing Abs following infection or traditional vaccination procedures.We reported previously the induction of nucleophilic Abs by covalent immunization with full-length gp120 and a gp120 V3 peptide containing strongly electrophilic phosphonate groups (
41–
43). The electrophile reacts covalently with BCRs (
44), resulting in adaptively strengthened nucleophilic reactivity coordinated with specific noncovalent recognition of gp120. The Abs obtained by covalent immunization formed very stable immune complexes with HIV resulting from pairing of Ab nucleophiles with the naturally occurring electrophilic groups of gp120 (
e.g. the backbone and side chain carbonyls, see Refs.
42,
43). A minority of the Abs proceeded to catalyze the hydrolysis of gp120, aided by water attack on the covalent acyl-Ab complex (
41). Here we report the neutralization of HIV strains heterologous to the full-length electrophilic gp120 immunogen (E-gp120) by mAbs with binary CD4BS and V3 loop recognition capability. We also present models that explain synthesis of the mAbs in response to immunization with E-gp120.
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