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Originally published In Press as doi:10.1074/jbc.M706471200 on August 29, 2007

J. Biol. Chem., Vol. 282, Issue 43, 31250-31256, October 26, 2007
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Towards Covalent Vaccination

IMPROVED POLYCLONAL HIV NEUTRALIZING ANTIBODY RESPONSE INDUCED BY AN ELECTROPHILIC gp120 V3 PEPTIDE ANALOG*

Yasuhiro Nishiyama{ddagger}1, Yukie Mitsuda{ddagger}, Hiroaki Taguchi{ddagger}, Stephanie Planque{ddagger}, Maria Salas§, Carl V. Hanson§, and Sudhir Paul{ddagger}2

From the {ddagger}Chemical Immunology Research Center, Department of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, Houston, Texas 77030 and the §Viral and Rickettsial Disease Laboratory, California Department of Public Health, Richmond, California 94804

Received for publication, August 6, 2007 , and in revised form, August 15, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain. Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain (MN) ~50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by gp120. Pretreatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies (Abs)3 that bind human immunodeficiency virus type 1 (HIV) with irreversible character are conceptually analogous to reagents with infinite binding affinity. Rare monoclonal Abs can form unusually stable immune complexes expressing covalent character (1, 2). The combining site of such monoclonal Abs is usually intended to replicate enzyme active sites. Immunization with an Ab to beta-lactamase, for example, is reported to induce an anti-idiotypic monoclonal Ab that forms a covalent intermediate with a beta-lactam compound that is sufficiently stable to be detected in denaturing electrophoresis gels (1). Monoclonal Abs raised to an analog of HIV gp120 containing electrophilic phosphonate diesters (E-gp120) form noncovalent immune complexes that are subsequently converted to irreversible complexes by nucleophile-electrophile interactions (2). In this example, stimulation with the electrophilic groups is suggested to strengthen the nucleophilic reactivity of Ab combining sites by the adaptive immunological processes that are also responsible for improved noncovalent binding, i.e. V-(D)-J gene recombination, somatic hypermutation and combinatorial diversification. B cell clonal selection is thought to be driven by binding of antigen to the B cell receptor (BCR), i.e. surface Ig complexed to signal transducing proteins. As irreversible binding should permit prolonged BCR occupancy, improvement of the nucleophilic reactivity over the course of adaptive B cell differentiation is feasible. The nucleophilic reactivity is reminiscent of enzymatic active sites, in which activated groups formed by intramolecular hydrogen bonding interactions acquire an ability to conduct nucleophilic attack at electrondeficient sites in substrates. For example, the nucleophilic reactivity of the Ser-His-Asp triad in serine proteases results in the formation of enzyme-substrate covalent reaction intermediates (3). Indeed, similar nucleophilic triads have been identified in monoclonal Ab combining sites by crystallography and mutagenesis studies (4–6). Completion of the catalytic cycle following the nucleophile-electrophile reaction requires various additional accessory groups in the active site. Consequently, some but not all nucleophilic Abs proceed to catalyze chemical reactions (7).

The ability to form long-lasting immune complexes can be anticipated to enhance the antigen inactivation potency of irreversibly binding Abs compared with their reversibly binding counterparts. This leads to the hypothesis that electrophilic antigen analogs can serve as the basis for vaccine formulations capable of inducing improved protective Ab responses to microbial antigens compared with conventional vaccines. The hypothetical improvement in vaccine formulation will depend on the proportion of the induced polyclonal Ab response that displays covalent character while maintaining the correct epitope specificity necessary for recognition of the native antigen structure. In previous studies using full-length E-gp120 as the immunogen, several monoclonal Abs were identified that formed unusually stable immune complexes with gp120 devoid of exogenously introduced electrophilic groups (2). However, full-length E-gp120 expresses a multitude of epitopes, and we were unable to relate Ab covalency and HIV neutralization because of the varying Ab epitope specificities. Moreover, a rigorous covalent ELISA protocol was employed to screen hybridoma supernatants. Consequently, there is no assurance that anything more than a small minority of the overall Ab response to E-gp120 expresses the desired covalent character of the monoclonal Abs.

The immune response following HIV infection is dominated by Abs to the principal neutralization determinant (PND) of gp120 corresponding to residues 306–328 located in the V3 domain (8–10). Immunization with the synthetic PND peptide induces Ab responses that neutralize HIV strains with sequences similar to the PND immunogen (11–14). Here, we studied the comparative HIV neutralizing and binding characteristics of polyclonal Ab preparations induced by an electrophilic analog of the PND peptide (E-PND) and the control PND peptide devoid of exogenously introduced electrophiles. We observed that immunization with E-PND induced Abs that neutralized HIV more potently and dissociated from intact HIV virions more slowly than control Abs from PND-immunized mice. These results indicate the improved antigen inactivation potency due to Ab nucleophilicity and suggest the utility of electrophilic immunization as a novel vaccination strategy.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
PND Analogs—PND peptide 1a corresponding to gp120 residues 306–328 of HIV strain MN (YNKRKRIHIGPGRAFYTTKNIIG) and its biotinylated analog (Bt-PND 1b) were prepared by Fmoc-based solid phase synthesis followed by purification with reversed phase HPLC (1a: observed m/z 2705.3; calcd m/z 2705.2. 1b: observed m/z 3043.0; calcd m/z 3044.6; Genemed Synthesis, South San Francisco, CA). The electrophilic phosphonate analog of PND (E-PND 2a) was prepared by acylation of 1a with N-hydroxysuccinimidyl ester of diphenyl (suberoyl) amino(4-amidinophenyl) methanephosphonate as follows. PND 1a (10 mg, 3.7 µmol) was allowed to react with the acylating agent (33 mg, 44 µmol) in dimethyl sulfoxide (6.6 ml) and 100 mM phosphate-buffered saline, pH 8 (4.0 ml) for 1 h. Excess acylating agent was quenched by addition of 1 M glycine (2 ml). 15 min thereafter, the precipitate was collected by centrifugation, washed with cold water (4 ml x 3), and subjected to HPLC purification (YMC-Pack ODS-AM, 4.6 x 250 mm; gradient of 10% to 80% acetonitrile in 0.05% trifluoroacetic acid/water, 45 min). This yielded chromatographically pure E-PND 2a (5.4 mg, 31%), which was characterized by electrospray ionization mass spectrometry (observed m/z, 1594.5, 1196.5, 957.7; calcd m/z for C235H319N51O50P4, 1595.1 (3+), 1196.6 (4+), 957.5 (5+)). Biotinylated E-PND (Bt-E-PND 2b) was prepared from Bt-PND 1b in the same manner (observed m/z, 1151.1, 921.3, 767.9; calcd m/z for C223H314N51O48P3S, 1151.6 (4+), 921.5 (5+), 768.0 (6+).

Antibodies—Female MRL/lpr mice (n = 5/immunogen; Jackson Laboratory, Bar Harbor, ME; 8 weeks age) were immunized intraperitoneally on days 0, 15, 29, 44, 58, and 101 with PND 1a or E-PND 2a (50 µg for the first 4 injections and 200 µg for the last 2 injections) in RIBI adjuvant (monophosphoryl lipid A-trehalose dicorynomycolate emulsion; Sigma-Aldrich). Blood was obtained from the retroorbital plexus over the course of the immunization schedule (days 0, 15, 29, 44, 58, 65, and 111). Development of PND-reactive IgG was examined by ELISA using Bt-PND 1b (4 µg/ml) immobilized on streptavidin-coated plates, sera diluted 1:5000 in 10 mM PBS containing 0.025% Tween 20 and 1% bovine serum albumin, and peroxidase-conjugated goat anti-mouse IgG (Fc specific; Sigma-Aldrich) as secondary Ab. IgG was purified to electrophoretic homogeneity from serum (prepared from blood collected 10 days after the last immunization) by affinity chromatography on protein-G Sepharose (GE Healthcare, Piscataway, NJ) (15).

HIV Neutralization—Neutralization of HIV (strain MN, clade B) by serially diluted sera or purified IgG samples was determined in a "microplaque" reduction assay using cells of the MT-2 T lymphocyte cell line as hosts (16). Neutralization of strain ZA009 (clade C) was measured by the p24 capsid protein assay with human peripheral blood mononuclear cells as hosts (17). Concentrations yielding 50 and 80% inhibition (IC50 and IC80) were obtained from the least-square-fits to a sigmoidal dose-response shown in Equation 1.

Formula 1(Eq. 1)

HIV Binding Assays—Purified IgG (17 µg/ml) and HIV (MN strain, 1.6 x 104 TCID50/ml; TCID50, 50% tissue culture infective dose) were incubated in a mixture of 10 mM phosphate-buffered saline, pH 7.4, and RPMI1640 (1:1) containing 10% fetal bovine serum at 4 °C for 16 h. HIV-IgG complexes (and free IgG) were captured on Protein G-Sepharose (100 µlof settled gel) using Bio-Spin chromatography columns (Bio-Rad), and unbound HIV removed by washing with the reaction buffer (500 µl x 8). The captured complexes were eluted with 100 mM glycine·HCl, pH 2.7 (400 µl), HIV was lysed with Triton X-100 (10%) and p24 in the lysates was measured with Coulter HIV-1 p24 Antigen Assay kit. Values were corrected for nonspecific HIV binding to the affinity gel, determined by identical processing of control HIV treated with diluent in the absence of IgG (A490, 0.21 ± 0.01). In competition experiments, IgG and HIV were incubated in the presence of excess PND peptide 1a (50 µg/ml) or an irrelevant peptide (gp120 residues 465–479 of MN strain; NIH AIDS Research and Reference Reagent program), and HIV-IgG complexes were measured as above. To determine dissociation kinetics, HIV-IgG complexes were allowed to form for 16 h (IgG 50 µg/ml, HIV 1.6 x 104 TCID50/ml), excess PND 1a (50 µg/ml) was added to the reaction mixtures to preclude reassociation of any complexes undergoing dissociation. Aliquots of the reaction mixtures withdrawn 0.5, 2, 4, 8, and 14 h thereafter were immediately subjected to protein G chromatography and p24 assays to determine residual immune complexes as described above.

IgG Nucleophilic Reactivity—Bt-E-PND 2b (1 µM) was incubated with IgG (75 µg/ml) for 2 h, the reaction mixtures were boiled, subjected to reducing SDS-electrophoresis, and the covalent adducts were detected by streptavidin-peroxidase staining of the blots as described previously (18). To study the role of IgG nucleophilic reactivity in HIV binding, the IgG (1.4 mg/ml) was treated with the haptenic phosphonate 3 (1 mM; diphenyl benzyloxycarbonylamino(4-amidinophenyl) methanephosphonate, synthesis described in ref 19) at room temperature for 13 h. After removing unreacted phosphonate by gel filtration (Bio-spin 6, Bio-Rad; IgG recovery 88%), the dissociation kinetics of the HIV-IgG complexes was studied as above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Immunogenicity—The E-PND immunogen 2a corresponds to the amino acid sequence of HIV gp120 residues 306–328 (MN strain) with 4 electrophilic phosphonate diester groups located at the side chains of Lys308, Lys310, and Lys324 residues and the N terminus (Fig. 1A). The phosphonate groups are structurally identical to those incorporated into full-length gp120 and other polypeptides in previous studies (7, 18, 20–22). The resultant electrophilic polypeptides bind nucleophilic sites in Abs covalently (18, 20–22). We employed multiple phosphonate groups within a single E-PND molecule to increase the probability of nucleophile-electrophile pairing coordinated with noncovalent binding at the proximate peptide regions. The control immunogen PND 1a was the identical peptide structure devoid of the phosphonate groups. Successive immunizations of mice with E-PND 2a or PND 1a resulted in progressively increasing PND binding activity in serum IgG obtained over days 15 to 65 (mean A490 ± S.D. for 1:5000-diluted pooled sera using immobilized Bt-PND 1b increased from 0.06 ± 0.10 to 0.71 ± 0.13 in 2a-immunized mice and from 0.01 ± 0.01 to 0.42 ± 0.28 in 1a-immunized mice; n = 5 mice each; preimmune mouse serum binding, 0.01 ± 0.01). ELISA assays using the final bleeds obtained on day 111 indicated that each of the mice had mounted an IgG response to PND (A490 values for sera from individual 2a-immunized mice at 1:1000 dilution, 0.79 ± 0.01, 0.47 ± 0.03, 1.48 ± 0.01, 0.72 ± 0.02, 2.59 ± 0.09). These results indicate that Abs to E-PND can recognize PND devoid of the electrophilic groups.

HIV Neutralization—Pooled sera from the mice (day 111) were assayed for the ability to neutralize HIV strain MN (clade B, coreceptor CXCR4-dependent) using MT-2 host cells. Serially diluted sera were incubated with the virus and the infectivity was measured by the microplaque assay (16). Dose-dependent HIV neutralization was observed, with the sera from E-PND 2a-immunized mice displaying 44–272-fold greater neutralizing potency compared with sera from PND 1a–immunized mice (dilution yielding 50% neutralization, 1:787 versus 1:34,444; dilution yielding 80% neutralization, 1:54 versus 1:14,692; Fig. 1B). The consistency of the Ab response was confirmed by assaying neutralization by sera from individual E-PND 2a-immunized mice and PND 1a-immunized mice (Fig. 1C; n = 5 and 4 mice, respectively). Mean % neutralization ± S.D. values at 1:800 dilution of sera from the E-PND and PND groups were, respectively, 92 ± 18 and 25 ± 2%; p = 0.0002, unpaired t test, two-tailed. The superior neutralizing Ab response in E-PND 2a immunization was confirmed using pooled IgG purified by affinity chromatography on protein G-Sepharose columns (Table 1). IgG from E-PND 2a-immunized mice was 59-fold more potent than IgG from PND 1a-immunized mice (IC50 values, respectively, 2 versus 118 µg/ml). Serum from E-PND or PND immunized mice at dilutions as low as 1:500 failed to neutralize the clade C strain ZA009 (core-ceptor R5-dependent; data not shown). This is consistent with the highly divergent sequences of the strain MN PND peptide employed as immunogen and the PND expressed by strain ZA009 (respectively, YNKRKRIHIGPGRAFYTTKNIIG and NNTRKSMRIGPGQVFYATNGIIG).


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  		TABLE 1
HIV neutralizing activity of anti-E-PND and anti-PND IgGs

Protein G-purified IgGs were assayed for HIV neutralization activity using the clade B X4 strain MN and MT-2 host cells. Concentrations yielding 50% and 80% neutralization (IC50 and IC80) were obtained from dose response studies (four replicates at each concentration), with curve fitting as in Ref. 17.

 


Figure 1
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  		FIGURE 1.
HIV neutralizing activity. A, immunogen structures. PND 1a is the synthetic peptide corresponding gp120 residues 306–328 (MN strain). E-PND 2a is an electrophilic analog of PND containing diphenyl amino(4-amidinophenyl) methanephosphonate groups (a) located at 3 Lys residues (Lys308, Lys310, Lys324) and the N terminus. PND 1b and E-PND 2b, respectively, are 1a and 2a analogs with biotinamidohexanoyl (b) at the N terminus. B, HIV neutralization by pooled sera from mice immunized with E-PND and PND. Serially diluted pooled sera (n = 5 mice) obtained following hyperimmunization with E-PND 2a or PND 1a (day 111) were assayed for HIV neutralization activity by the microplaque assay using the clade B, CXCR4-dependent strain MN and MT-2 host cells (values are means ± S.E. of 4 replicates at each concentration studied in parallel). Number of plaques in the absence of sera was 27.7 ± 1.7/well. C, HIV neutralization by sera from individual mice immunized with E-PND 2a or PND 1a. Sera from the mice (day 111; 1:800 dilution) were assayed for HIV neutralization activity as in panel B (values are means ± S.E. of 4 replicates conducted in parallel). Number of plaques in the absence of sera was 24.8 ± 1.2/well.

 


Figure 2
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  		FIGURE 2.
Intact HIV-IgG binding. A, HIV binding by anti-E-PND IgG and anti-PND IgG. IgG (17 µg/ml) and HIV (MN, 1.6 x 104 TCID50/ml) were incubated at 4 °C for 16 h. Formation of HIV-IgG immune complexes was measured by capture on immobilized protein G and p24 assays (see "Experimental Procedures"). Values are means ± S.D. of duplicates corrected for nonspecific binding, determined by identical processing of HIV in the absence of IgG (A490, 0.210 ± 0.005). B, inhibition of HIV-IgG binding by PND peptide 1a. IgG (50 µg/ml) and HIV (MN, 1.6 x 104 TCID50/ml) were incubated in the presence (50 µg/ml) of the PND peptide or an irrelevant peptide control (gp120 465–479), and IgG-bound HIV was determined as in panel A. Values are expressed as percent of binding observed in the absence of 1a (A490 0.49 ± 0.02, anti-PND IgG; A490 0.47 ± 0.01, anti-E-PND IgG). Shown is a representative assay of three independent assays. Residual HIV binding in the presence of 1a in these assays were 9 ± 4% for anti-PND IgG (A490 in the absence of 1a, 0.61 ± 0.09) and 12 ± 3% for anti-E-PND IgG (0.62 ± 0.12).

 
HIV Binding Characteristics—To study Ab binding to the native PND structure expressed on the viral surface, we used intact, infectious HIV particles. The virion preparation (MN strain) was incubated with purified IgG, the HIV-IgG complexes were captured using immobilized protein G, the complexes were eluted at acid pH and virions were lysed with a detergent, and p24 was measured by ELISA. As shed gp120 is not associated with p24, the method detects Ab-HIV complexes without interference from free gp120 shed by the virus. Binding of the virions by IgG preparations from E-PND 2a-immunized and PND 1a-immunized mice was evident at levels greater than control IgG from preimmune mice (Fig. 2A). Near-complete competitive inhibition of the binding was observed in the presence of excess PND 1a but not an irrelevant HIV peptide (Fig. 2B), indicating that virion binding by IgG induced by E-PND 2a immunization is attributable to specific recognition of the PND region.

To study immune complex stability, we measured the dissociation rates of the HIV-IgG complexes. After the complexes had been formed, excess PND 1a (18.5 µM) was added to the reaction mixtures to preclude reassociation of HIV that had undergone dissociation, and the residual complexes were measured periodically by the p24 assay. Dissociation of HIV complexed to IgG induced by immunization with PND 1a occurred rapidly and proceeded at a rate consistent with the first-order dissociation equation (half-life, t1/21, 10.6 min; r2 0.985; Fig. 3). Dissociation of anti-E-PND IgG complexes was substantially slower and suggested two subpopulations of complexes, a subpopulation that dissociated slowly over 8 h (~70% of initial complexes; nominal t1/21 assuming first order kinetics and complete dissociation over 8 h, 2.5 h, r2 0.971) and another subpopulation that did not dissociate detectably between 8 and 14 h (~30% of initial complexes).


Figure 3
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  		FIGURE 3.
Dissociation of HIV-IgG complexes. Immune complexes were formed by incubating anti-E-PND or anti-PND IgG (50 µg/ml) with HIV (MN, 1.6 x 104 TCID50/ml) for 13 h. PND peptide 1a (50 µg/ml) was added (t = 0), and aliquots of the mixtures withdrawn at the indicated time points were processed for measurement of residual HIV-IgG complexes as in Fig. 2. Values are expressed as percent of binding at t = 0 (100% A490 values for anti-E-PND and anti-PND IgGs, respectively: 0.56 ± 0.01 and 0.37 ± 0.03; corrected for nonspecific binding determined using HIV-IgG mixtures in which 50 µg/ml PND peptide was present before initiating immune complex formation; values of nonspecific binding with anti-E-PND and anti-PND IgGs, respectively: 0.15 ± 0.01 and 0.16 ± 0.02). Shown is one of two independent assays (nominal t1/21 values of HIV complexes formed by anti-PND and anti-E-PND IgGs in the assay not shown here were 12 min and 2.1 h, respectively).

 
Ab Nucleophilic Reactivity—The strongly electrophilic phosphonate in probe E-PND 2b (Fig. 1A) is known to form stable covalent bonds with Ab nucleophiles (7, 18, 20–22). In addition, this probe contains the PND peptide regions available for noncovalent Ab binding. As a test of antigen-specific nucleophilic reactivity, we measured the formation of covalent adducts of IgG and Bt-E-PND 2b. Boiled reaction mixtures of the IgG and E-PND 2b were analyzed by reducing SDS-electrophoresis and densitometry. Adducts of 2b formed by the IgG heavy and light chains from E-PND-immunized mice accumulated more rapidly than the adducts from control PND-immunized mice or control non-immune IgG (Fig. 4A, 60 kDa heavy chain adduct intensities in arbitrary volume units from 2a-immunized mice, 1a-immunized mice and preimmune mice, respectively, 3881, 830, and 111; 30-kDa light chain adduct intensities, respectively, 1869, <12, and <12). In previous studies, we employed the small molecule phosphonate 3 (Fig. 4B) as a probe for nucleophilic reactivity of Abs independent of noncovalent binding forces responsible for specific recognition of polypeptide antigens (18, 19, 21, 23). According to our split-site model deduced from mutagenesis studies (5, 24, 25), distinct sites in antigen-specific Abs are responsible for the nucleophilic and noncovalent binding activities. Following treatment with excess phosphonate 3 and removal of the unreacted phosphonate 3, IgG from E-PND immunized mice retained the ability to bind HIV. However, the dissociation of HIV complexes formed by phosphonate 3-treated anti-E-PND IgG was accelerated compared with the control IgG treated with diluent, and unlike the control HIV-IgG complexes, the dissociation of HIV complexes formed by phosphonate 3-treated IgG proceeded to near-completion within 1 h (Fig. 4B). These results suggest that nucleophilic sites in IgG from the E-PND-immunized mice are responsible for the enhanced stability of the immune complexes and superior HIV neutralization.


Figure 4
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  		FIGURE 4.
IgG nucleophilic reactivity. A, formation of IgG adducts with E-PND 2b. The anti-E-PND IgG, anti-PND IgG, and preimmune IgG (75 µg/ml) were incubated with E-PND 2b (1 µM) for 2 h, subjected to reducing SDS-electrophoresis and the covalent adducts were detected by streptavidin-peroxidase staining of blots. B, dissociation of HIV complexes formed by hapten 3-treated IgG. Anti-E-PND IgG (1.4 mg/ml) was incubated in the presence of diluent or hapten phosphonate 3 (1 mM; structure in inset) for 16 h, and unreacted 3 was removed by gel filtration. Immune complexes were formed by incubating 3-treated anti-E-PND IgG or control diluent-treated anti-E-PND IgG (50 µg/ml) with HIV (MN, 1.6 x 104 TCID50/ml) for 13 h. Dissociation of HIV-IgG complexes (means ± S.D., three replicates at each time point) were monitored periodically as in Fig. 3. Values are corrected for nonspecific binding and are expressed as percent of binding at t = 0 (100% values for 3-treated IgG and diluent-treated control IgG, respectively: 0.73 ± 0.06 and 0.88 ± 0.03). C, likely mechanism for stabilization of immune complexes. The irreversible binding character of the immune complexes may be due to accumulation of the resonant tetrahedral complex (IC'). In enzymes, such complexes proceed to form trigonal acyl-enzyme covalent intermediates with release of the C-terminal antigen fragment. NuH, nucleophile; Ag1 and Ag2 denote flanking regions of the antigen responsible for noncovalent binding.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
An effective vaccine against diverse HIV strains found in different geographical regions and escape mutants that emerge in the course of infection remains elusive. The anti-viral efficacy of various Abs directed to the neutralizing epitopes of the HIV envelope depends largely on their first order dissociation rate constants (koff; t1/21 = ln 2/koff) and concentrations in biological fluids. The Ab-virus association rate constant (kon) also contributes to the observed binding affinity (Ka = kon/koff), but different Abs to a given viral epitope usually display comparable kon values, as this constant is controlled mainly by the rate of diffusion and the orientation of collisions between the reactants. A previous study has highlighted the correlation between the HIV neutralizing potency and dissociation rate constants of monoclonal Abs to the PND, the epitope targeted in the present study (26). We reported that rare monoclonal Abs raised to an electrophilic analog of full-length gp120 neutralized HIV and formed unusually stable immune complexes with gp120 devoid of exogenous electrophiles (2). The stability of the immune complexes was attributable to the adaptively strengthened nucleophilic groups in the Abs induced by electrophilic immunization. The nucleophilic groups are hypothesized to lend covalent character to the complexes by pairing with natural electrophilic groups in gp120. Regrettably, polyclonal IgG from the mice immunized with full-length E-gp120 failed to neutralize HIV at levels superior to IgG from non-immunized mice, presumably because the overall polyclonal immune response is dominated by Abs to irrelevant epitopes and Abs with sufficient nucleophilic reactivity to the neutralizing epitope were present only at low concentrations.

In the present study, a well characterized synthetic analog of the peptide corresponding to the principal neutralizing determinant of HIV strain MN was studied as immunogen (E-PND). Four electrophilic phosphonates were incorporated within the 23 residues peptide analog to maximize the opportunity of B cell adaptive differentiation in response to electrophilic stimulation. Polyclonal Ab preparations obtained following E-PND immunization formed complexes with intact HIV virions. The complexes were poorly dissociable or not at all dissociable. The polyclonal anti-E-PND IgG neutralized HIV ~50-fold more potently than control Abs to PND devoid of phosphonate electrophilic groups. This is consistent with the prediction that slower dissociation of the HIV-IgG complexes should prolong the duration over which the Ab-complexed virus exists in noninfectious form. Biochemical analysis confirmed the enhanced nucleophilic reactivity of the anti-E-PND Abs and the importance of the nucleophilic reactivity in prolonging immune complex longevity. It may be concluded that the nucleophilic Abs responsible for forming stable immune complexes are present in the polyclonal IgG mixtures at concentrations sufficient to achieve functionally useful viral inactivation.

Concerning epitope specificity, the PND (residues 306–328) is located in the highly mutable V3 region of gp120 (12). Consistent with the neutralization results for strains MN and ZA009 in the present study, Abs to the V3 region display type-specific neutralizing activity, that is, they neutralize the infecting HIV strain, but V3 sequence mutants resistant to infection emerge following infection (11–14). The mechanism of neutralization by anti-PND Abs is thought to involve sterically hindered recognition of gp120 by HIV coreceptors CCR5 and CXCR4 expressed by host cells (27–29). In particular, residues Pro316-Arg318 are reported to be important for CCR5 binding (30–32). Attempts to induce broadly neutralizing Abs that recognize diverse HIV strains using mixtures of synthetic peptides with varying V3 sequences have been reported (e.g. Refs. 33, 34). However, V3 sequence diversity is so great that a large library of immunogenic peptides is necessary to justify hopes of inducing a broadly neutralizing Ab response. For example, the 201 known clade B virus strains available in the Los Alamos data base contain 179 distinct PND sequences. Monoclonal Abs that recognize the more conserved Gly-Pro-Gly-Arg sequence within the PND are reported to neutralize various HIV strains comparatively broadly (35, 36). To the extent that inducing a broadly neutralizing Ab response to the PND or regions within the PND is feasible by these means, our results suggest that inclusion of electrophilic groups in the immunogenic peptide (or combination of peptides) will be helpful to increase the potency of neutralization. In principle, the electrophilic immunization strategy is applicable to targeting of any peptide epitope. gp120 also contains important neutralizing epitopes outside the PND, e.g. the conserved regions that participate in binding to host cell CD4 receptors (37). However, these epitopes are generally poorly immunogenic and they are composed of peptide regions distant in the linear sequence of the protein (conformational epitopes). No linear gp120 peptide or mimetic is available presently that reproducibly induces the synthesis of broadly neutralizing Abs to the diverse HIV strains responsible for the pandemic.

The following empirical and theoretical points are relevant in assessing the potential generality of the electrophilic immunization approach: (a) Electrophilic phosphonates were originally developed as covalent inhibitors of the catalytic sites of serine proteases (38). Haptenic phosphonates react covalently with all Ab preparations examined thus far, including monoclonal Abs, single chain Fv constructs (VL and VH domains tethered by a linker) (18, 19, 23) and Igs contained in BCRs (39). This suggests that the nucleophilic reactivity is ubiquitously distributed in Ab combining sites regardless of noncovalent binding specificity. (b) Noncovalent epitope recognition is reported to accelerate the covalent reaction of electrophilic phosphonate groups incorporated into several polypeptide with various polyclonal and monoclonal Abs specific for these polypeptides (18, 20–22). It appears, therefore, that the noncovalent and nucleophilic Ab subsites are within sufficient proximity to express their functions in a coordinated manner. (c) Only a small subset of nucleophilic Abs displays the ability to hydrolyze peptide bonds (7). This may be understood from the requirement for additional rate-limiting events for completing the catalytic cycle following the initial nucleophilic attack step, i.e. water attack and product release. We did not detect PND hydrolysis IgG from E-PND immunized mice.4 Importantly, the electrophilic phosphonate is predicted to favor adaptive strengthening of Ab nucleophilicity, but the immunogen lacks structural elements that can induce the synthesis of Abs capable of completing the catalytic cycle; and (d) Observations using noncatalytic monoclonal Abs raised to full-length E-gp120 suggest the feasibility of immune complex stabilization by resonant nucleophile-electrophile pairing at the naturally occurring electrophilic reaction centers in the polypeptide antigen, e.g. the carbonyl groups of backbone peptide bonds or side chain amide bonds in structure IC', Fig. 4C (2). Such interactions are hypothesized to impart partial covalent character to the complexes and impede their dissociation. Similar structures are thought to exist in the transition state of enzymatic reactions (3). To our knowledge there is no theoretical bar to stabilization of ground state protein-protein complexes by this mechanism. B cell clonal selection processes favor increased BCR occupancy by the antigen. Covalent binding of electrophilic phosphonates due to improved BCR nucleophilic reactivity is predicted to be an immunological selectable event, and the desirable consequence of the improved nucleophilicity is the ability to bind the target polypeptide antigen with covalent character.

In summary, our studies using the model E-PND immunogen indicate that electrophilic immunization induces a robust polyclonal nucleophilic Ab response with improved viral binding and inactivation potency. Concerning HIV vaccine development, the caveat remains that PND sequence divergences may limit the functional efficacy of E-PND immunization. However, the E-PND studies validate electrophilic immunization as a potentially general approach that can be applied to induce adaptive Ab responses capable of binding microbial antigens with irreversible character and help improve vaccine efficacy.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants, R01AI058865, R01AI067020, R21AI062455, and R21AI071951. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed: Chemical Immunology Research Center, Dept. of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030. E-mail: Yasuhiro.Nishiyama{at}uth.tmc.edu. 2 To whom correspondence may be addressed: Chemical Immunology Research Center, Dept. of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030. E-mail: Sudhir.Paul{at}uth.tmc.edu.

3 The abbreviations used are: Ab, antibody; Bt-, biotinamidohexanoyl; BCR, B cell receptor; E-gp120, gp120 analog containing electrophilic phosphonates; E-PND, principal neutralizing determinant analog containing electrophilic phosphonates; HIV, human immunodeficiency virus; PND, principal neutralizing determinant; TCID50, 50% tissue culture infective dose. Back

4 Incubation of PND 1a (0.1 mM) in the presence of IgG (1 µM) from hyperimmune mice for 3 h did not result in detectable product accumulation at levels greater than background in control incubations conducted using an equivalent concentration of preimmune IgG determined by reversed-phase HPLC with an electrospray mass detector (VyDac C18 MASS SPEC column, 2.1 x 150 mm; 3–60% acetonitrile in 0.1% formic acid and water; retention time of intact PND 1a, 14.3 min). Back


   ACKNOWLEDGMENTS
 
We thank Robert Dannenbring and Tomoko Yoshikawa for technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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