JOURNAL OF VIROLOGY, Oct. 2005, p. 12311–12320 0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.19.12311–12320.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 19
Kinetic Rates of Antibody Binding Correlate with Neutralization Sensitivity of Variant Simian Immunodeficiency Virus Strains† Jonathan D. Steckbeck,1 Irina Orlov,3 Andrew Chow,3 Heather Grieser,1 Kenneth Miller,3 JoAnne Bruno,3 James E. Robinson,4 Ronald C. Montelaro,2 and Kelly Stefano Cole1* Department of Medicine, Infectious Diseases Division,1 and Department of Molecular Genetics and Biochemistry,2 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Biacore, Inc., Piscataway, New Jersey 088543; and Department of Pediatrics, Tulane Medical Center, New Orleans, Louisiana 701124 Received 22 October 2004/Accepted 28 May 2005
Increasing evidence suggests that an effective AIDS vaccine will need to elicit both broadly reactive humoral and cellular immune responses. Potent and cross-reactive neutralization of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) by polyclonal and monoclonal antibodies is well documented. However, the mechanisms of antibody-mediated neutralization have not been defined. The current study was designed to determine whether the specificity and quantitative properties of antibody binding to SIV envelope proteins correlate with neutralization. Using a panel of rhesus monoclonal antibodies previously characterized for their ability to bind and neutralize variant SIVs, we compared the kinetic rates and affinity of antibody binding to soluble envelope trimers by using surface plasmon resonance. We identified significant differences in the kinetic rates but not the affinity of monoclonal antibody binding to the neutralization-sensitive SIV/17E-CL and neutralization-resistant SIVmac239 envelope proteins that correlated with the neutralization sensitivities of the corresponding virus strains. These results suggest for the first time that neutralization resistance may be related to quantitative differences in the rates but not the affinity of the antibody-envelope interaction and may provide one mechanism for the inherent resistance of SIVmac239 to neutralization in vitro. Further, we provide evidence that factors in addition to antibody binding, such as epitope specificity, contribute to the mechanisms of neutralization of SIV/17E-CL in vitro. This study will impact the method by which HIV/SIV vaccines are evaluated and will influence the design of candidate AIDS vaccines capable of eliciting effective neutralizing antibody responses. Increasing evidence for the role of neutralizing antibodies in protective immunity comes from passive serum/antibody experiments that demonstrate the efficacy of antibodies against viral exposure in the human immunodeficiency virus type 1 (HIV-1)/chimp (11, 37), simian immunodeficiency virus (SIV)/ monkey (8, 20, 21, 49, 50), and simian/human immunodeficiency virus (SHIV)/monkey (2, 18, 31–34, 47) systems. Evidence for cross-reactive neutralizing antibody responses in HIV-1-infected long-term nonprogressors suggests that antibody may also be involved in the control of virus infection and prevention of disease progression (4). In support of this, we previously demonstrated the emergence of broadly neutralizing antibodies in SIV-infected macaques at a time when antibody maturation and protection against pathogenic challenge have been achieved (8, 10, 36). These studies demonstrate the ability of neutralizing antibodies to mediate protection against pathogenic challenge in the absence of other immune responses and suggest a role for neutralizing antibody responses during the chronic stage of infection. Thus, candidate AIDS
* Corresponding author. Mailing address: University of Pittsburgh School of Medicine, Department of Medicine, Infectious Diseases Division, 3550 Terrace Street, Scaife Hall, Suite 867, Pittsburgh, PA 15261. Phone: (412) 648-8583. Fax: (412) 648-8455. E-mail:
[email protected]. † Supplemental material for this article may be found at http://jvi .asm.org/.
vaccines must be designed to elicit potent neutralizing antibody responses. Neutralizing antibodies act, in part, by blocking binding and/or entry of virus into permissive cells. However, the mechanism(s) by which antibody-mediated neutralization of virus occurs is still poorly defined. One hypothesis is that neutralization sensitivity is related to the density of envelope spikes on the virion, requiring a threshold level of antibody binding to limit infectivity (27, 38–40). Alternatively, neutralization of virus by antibody has been associated with differences in the qualitative antibody binding properties, i.e., antibody affinity and epitope specificity (9, 15, 26, 56). Previous studies have suggested a relationship between antibody affinity for oligomeric gp120 and neutralization of HIV-1 (19, 44, 46), and monoclonal antibodies (MAbs) that bind the V3 loop of diverse HIV-1 gp120 proteins with similar association rates exhibited marked differences in the dissociation rates that were predictive of neutralization capacity (48). However, antibody binding alone may not be sufficient to predict neutralization, since high-titer, high-affinity nonneutralizing antibody responses to both SIV and HIV-1 envelope proteins have also been identified (26, 56). In addition, neutralization may involve antibody recognition of epitopes that are not readily accessible on the native envelope trimer (15, 17, 56). Together, these data suggest that a combination of qualitative antibody binding properties and epitope specificity is likely to be involved in neutralization. Understanding the mechanism(s) of antibody-
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mediated neutralization will facilitate the design and evaluation of candidate AIDS vaccines. We have developed a panel of rhesus MAbs derived from monkeys infected with attenuated SIV/17E-CL that identify at least eight binding domains on gp120 (9, 45). This panel of MAbs represents both linear and conformational binding domains, including three groups of neutralizing MAbs that recognize discontinuous epitopes in the C-terminal half of SIV gp120. The latter three groups of MAbs neutralize the homologous SIV/17E-CL virus and a heterologous primary isolate, SIVdeltaB670, but fail to neutralize SIVmac239 in vitro. The main goal of the current study was to determine, by using surface plasmon resonance (SPR) analyses, whether qualitative properties of rhesus MAb binding to SIV envelope proteins could account for observed differences in neutralization of viruses in vitro. Results from these studies provide kinetic evaluations of MAb binding to diverse epitopes in the viral envelope protein relevant to neutralization and, for the first time, demonstrate that differences in the kinetic rates of antibody binding to recombinant SIV envelope correlate with the neutralization sensitivities of the corresponding virus strains. MATERIALS AND METHODS Rhesus MAb production and purification. Rhesus MAbs were produced from rhesus Epstein-Barr virus-immortalized cell lines and purified from harvested cell culture supernatants on protein A-Sepharose columns (Sigma, St. Louis, MO) as previously described (9, 45). Virus production. SIVmac239 and SIV/17E-CL virus stocks were prepared by transfection of CEMx174 cells in 75-cm2 culture flasks. Supernatant fluids were collected 1 week after transfection, filtered, aliquoted, and stored at ⫺80°C. The infectious titer of each virus stock was determined by incubating 10-fold dilutions of culture supernatants containing SIVmac239 or SIV/17E-CL with Tzm-bl cells for 48 h at 37°C. Tzm-bl cells are a HeLa cell line engineered to express human CD4, CCR5, and CXCR4. In addition, luciferase expression is driven by the HIV-1 long terminal repeat upon infection of the cells with HIV-1 or SIV strains (52). Following infection, supernatants were removed, and the cells were washed gently with phosphate-buffered saline (PBS) and lysed in 50 l lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N⬘,N⬘-tetraacetic acid, 10% glycerol, 1% Triton X-100) for 15 min at room temperature. Cell lysates (25 l) were transferred to a white luminescence plate (USA Scientific, Ocala, FL), 20 l luciferase substrate (Promega, Madison, WI) was added to each well, and the samples were assayed for luciferase production using a Lmax II luminometer (Molecular Devices, Sunnyvale, CA). The 50% tissue culture infective dose for each virus stock was determined using the method of Reed and Muench (41). Virus neutralization. Virus neutralization assays were performed in 96-well tissue culture plates by determining the concentration of MAb capable of reducing a constant amount of virus (multiplicity of infection, 0.01) in Tzm-bl cells by 50% (35). Serial fivefold dilutions of each rhesus MAb or medium alone were incubated with virus diluted to a multiplicity of infection of 0.01 for 1 h at 37°C. The virus-MAb mixtures were added to Tzm-bl cells (104 cells/well) and incubated for 48 h at 37°C. Following infection, supernatants were removed, and the cells were washed gently with PBS and lysed in 50 l lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexaneN,N,N⬘,N⬘-tetraacetic acid, 10% glycerol, 1% Triton X-100) for 15 min at room temperature. Cell lysates (25 l) were transferred to a white luminescence plate (USA Scientific, Ocala, FL), 20 l luciferase substrate (Promega, Madison, WI) was added to each well, and the samples were assayed for luciferase production using a Lmax II luminometer (Molecular Devices, Sunnyvale, CA). The 50% infectivity titers were determined using the method of Karber (25). Neutralizing titers are reported as the concentration (g/ml) of antibody that neutralized 50% of the virus infection, and the values reported represent averages of at least three independent experiments. Recombinant envelope protein production and purification. SIVmac239 (43) and SIV/17E-CL (1) recombinant gp140 (rgp140) (gp120 with the ectodomain of gp41) proteins were expressed and produced from vaccinia virus infection of 293T cells as previously described (5, 16). Briefly, the envelope gene from
J. VIROL. SIV/17E-CL was cloned into pSC65 (kindly provided by Bernard Moss, National Institutes of Health [NIH], Bethesda, MD), and a premature stop codon just N-terminal to the membrane-spanning domain was introduced using PCR-based mutagenesis. Recombinant vaccinia viruses were made by using these plasmids and the Western Reserve vaccinia virus strain using standard techniques (14). The virus containing SIVmac239 envelope (vCB74) was produced using the same protocol and was kindly provided by Robert Doms (University of Pennsylvania, Philadelphia, PA) (16). Recombinant vaccinia viruses were used to infect 293T cells at a multiplicity of infection of 10. The secreted envelope proteins were harvested as cell culture supernatants and purified by lentil lectin chromatography using 0.5 M methyl-␣-D-mannopyranoside (Sigma, St. Louis, MO) for elution as previously described (5). ConA ELISA. We determined the reactivities of rhesus MAbs to SIV rgp140 envelope proteins by a concanavalin A (ConA) enzyme-linked immunosorbent assay (ELISA) as previously described (9). Briefly, rgp140 proteins (0.1 g/well) were captured for 1 h at room temperature on Immulon 2HB microtiter plates (Thermo Electron Corp., Waltham, MA) coated with ConA. Following a wash with PBS, all wells were blocked by the addition of 5% dry milk in PBS (blotto) for 1 h at room temperature. Rhesus MAbs were diluted in blotto and incubated in the SIV envelope-coated wells for 1 h at room temperature. Following extensive washing, peroxidase-conjugated anti-monkey immunoglobulin G (IgG) (Nordic Immunology Laboratories, Tilburg, The Netherlands) was diluted in blotto, added to each well, and incubated for 1 h at room temperature. After a final washing step, all wells were incubated with TM Blue substrate (Serologicals Corp., Norcross, GA) for 20 min at room temperature, color was developed by the addition of 1 N sulfuric acid, and the wells were read (optical density [OD] at 450 nm) using an automated ELISA plate reader (Thermo Electron Corp., Waltham, MA). Relative affinity is reported as the concentration at half-maximal MAb binding. CD4 binding ELISA. The reactivities of viral or recombinant gp140 SIVmac239 and SIV/17E-CL envelope antigens to recombinant soluble CD4 (rsCD4) were determined by a ConA ELISA. Envelope proteins were captured on ConA-coated plates as described above. All wells were blocked with 5% dry milk in PBS before being incubated with 10 ng/well rsCD4 (kindly provided by the AIDS Research and Reference Reagent Program, NIH, Rockville, MD) for 1 h at room temperature. All wells were incubated with a 1:1,000 dilution of a rabbit anti-CD4 antibody (kindly provided by the AIDS Research and Reference Reagent Program) for 1 h at room temperature, followed by incubation with peroxidase-labeled goat anti-rabbit IgG (Nordic Immunology) for 1 h at room temperature. TM Blue substrate (Serologicals Corporation, Gaithersburg, MD) was added to each well for 20 min at room temperature, color was developed by the addition of 1 N sulfuric acid, and the OD at 450 nm was read using an automated ELISA plate reader (Dynex Technologies, Chantilly, VA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses. Viral or recombinant envelope proteins were resolved on a Criterion 4 to 15% continuous-gradient polyacrylamide gel or a Criterion XT Tris-acetate 3 to 8% continuous-gradient polyacrylamide gel (both from Bio-Rad, Hercules, CA) before being transferred to polyvinylidene difluoride membranes. Viral proteins were detected using polyclonal SIV Ig or monoclonal antibodies specific for Gag (2F12; kindly provided by the AIDS Research and Reference Reagent Program) or envelope (3.11H) (9). To quantitate envelope proteins present in the different virus stocks, virus was first pelleted over a glycerol cushion at 19,000 ⫻ g for 2 h at 4°C and resuspended in PBS. Following resolution on a 4 to 15% continuous-gradient polyacrylamide gel (Bio-Rad, Hercules, CA), viral proteins were transferred to a polyvinylidene difluoride membrane and probed with MAbs 2G12 and 3.11H. Densitometry analyses were performed, and the ratios of Gag to envelope were determined in order to compare the content of envelope between viruses. SPR analysis. We performed kinetic analyses of rhesus MAb binding to SIV rgp140 on a Biacore 3000 (Biacore AB, Inc., Uppsala, Sweden). Protein A (Pierce, Rockford, IL) was covalently bound to individual flow cell surfaces of a CM5 sensor chip with amine-coupling chemistry. Rhesus MAbs were captured and oriented onto protein A surfaces to ensure that the MAb-envelope binding occurred as a homogenous 1:1 Langmuir interaction. SIV rgp140 was injected over each flow cell, and each experiment analyzed MAb-rgp140 interactions at concentrations of the rgp140 trimer ranging from 0.27 to 133 nM. A buffer injection served as a negative control. All experiments contained an additional protein A control surface that served to account for changes in the buffer refractive index and to test for potential nonspecific interactions between rgp140 and protein A. Upon completion of each association and dissociation cycle, surfaces were pulsed with regeneration solution. Rhesus MAb association rates (ka), dissociation rates (kd), and affinity constants (KD) were calculated with BIAevaluation 4.1 software (Biacore AB, Uppsala, Sweden). The goodness of
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TABLE 1. Neutralizing antibody titers for SIV MAbs MAb
3.8E 3.10A 5.5B 3.11H 1.11A 4.7A 3.11E 3.2C E31 1.10A C26 3B3
Neutralizing antibody titer (g/ml)bfor:
Binding groupa
I II III IV V V VIa VIb VIb VII VII VIII
SIV/17E-CL
SIVmac239
⬎25 ⬎25 ⬎25 0.070 0.125 0.021 0.038 0.015 0.039 0.009 0.006 ⬎25
⬎25 ⬎25 ⬎25 ⬎25 ⬎25 ⬎25 ⬎25 ⬎25 ⬎25 ⬎25 ⬎25 ⬎25
a
Groups were previously defined by cross-competition ELISA (9). Neutralizing antibody titers are reported as the amount of antibody required to inhibit 50% infection in the Tzm-bl assay; values from a standard assay measuring cytopathic effect have been reported previously (9). b
each fit was based on the agreement between experimental data and the calculated fits, where the 2 values were below 1.0. Protein A-MAb surface densities were optimized to minimize mass transfer, and kinetic analyses demonstrated that the binding interactions were not significantly mass transfer limited. Statistical analysis. Statistical analyses were performed using InStat statistical analysis software (GraphPad, Inc., San Diego, CA). We determined the significance of the difference between paired MAb binding to SIVmac239 and SIV/ 17E-CL rgp140 proteins by using the Wilcoxon signed-rank test. The significance of the difference between neutralizing and nonneutralizing MAb reactivities to SIV/17E-CL rgp140 was determined using the Mann-Whitney test.
RESULTS Rhesus MAbs effectively neutralize SIV/17E-CL but not SIVmac239. We previously generated a panel of rhesus MAbs from four monkeys infected with attenuated SIV/17E-CL. Characterization of these MAbs included neutralization studies, where we identified three competition groups of MAbs that effectively neutralized SIV/17E-CL by using a standard neutralization assay where dilutions of antibody were incubated with a constant amount of virus and the ability of antibodies to inhibit SIV infection was determined using a readout of cytopathic effect 14 days postinfection (9). We have recently optimized a more-sensitive quantitative neutralization assay using Tzm-bl cells. Tzm-bl cells (52) are a HeLa cell line engineered to express human CD4, CCR5, and CXCR4. In addition, luciferase expression is driven by the HIV-1 long terminal repeat upon infection of the cells with HIV-1 or SIV strains. Thus, neutralization studies using this cell line are quantitative and reproducible, are performed in 48 h, and can be engineered to quantitate a single cycle of infection. In this study, we assayed the ability of a subset of these rhesus MAbs to neutralize SIV/17E-CL and SIVmac239 by using Tzm-bl cells. Results from these neutralization studies are summarized in Table 1. As shown previously, rhesus MAbs in competition groups V, VI, and VII neutralized SIV/17E-CL at concentrations of antibody similar to those previously reported using a standard neutralization assay (9). In addition, in the current study we also observed neutralization with the V3-specific MAb 3.11H, a result not observed with the standard neutral-
FIG. 1. Quantitation of envelope in SIV/17E-CL and SIVmac239 virions. Virus particles were pelleted from cell culture supernatants and subjected to polyacrylamide gel electrophoresis under denaturing conditions prior to analysis by Western blotting. Reactivity with antibodies specific for Gag (p27) and envelope (gp120) demonstrated similar ratios of Gag to envelope for the two viruses.
ization assay. Finally, no neutralization of SIVmac239 was observed. Envelope content does not account for differences in neutralization sensitivity between SIV/17E-CL and SIVmac239. Recent reports have suggested that sensitivity to neutralization may be related to the density of envelope on the virion surface, where larger amounts of envelope may be related to increased resistance to neutralization (29, 30, 51, 53). While the majority of the studies published to date described increases in envelope content on the cell or virion surface following truncations or mutations in gp41, no one has analyzed the potential differences in envelope content as they may be related to discrete differences in gp120. Since SIV/17E-CL and SIVmac239 differ by only 10 amino acids in gp120 yet demonstrate markedly different sensitivities to neutralization in vitro, we were interested in comparing the envelope content of SIV/17E-CL with that of SIVmac239. To address this question, equal amounts of pelleted viruses were analyzed by Western blot analyses using monoclonal antibodies specific for SIV Gag and envelope. As shown in Fig. 1 and Table 2, the ratios of Gag (p27) to envelope (gp120) were similar for SIV/17E-CL and SIVmac239. These results suggested that the dramatic differences in neutralization sensitivity observed between the two virus strains could not be attributed to differences in envelope content present on the surfaces of the virions. Rhesus MAbs bind envelope proteins from both neutralization-sensitive and neutralization-resistant SIV in ELISAs. Classical assays to measure antibody binding to a given antigen include Western blot analysis and ELISA. We previously reported the ability of a panel of rhesus MAbs to bind homologous SIV/17E-CL gp120 proteins in nonreducing Western
TABLE 2. Comparison of envelope content on SIV/17E-CL and SIVmac239 particles Reactivity (OD/mm2) SIV/17E-CL
SIVmac239
Ratio (SIV/17E-CL to SIVmac239)
3.58 4.45
2.36 2.82
1.52 1.57
Protein
Gag (p27) Env (gp120)
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FIG. 2. Characterization of SIV rgp140 trimers. (A) Recombinant gp140 proteins from SIV/17E-CL and SIVmac239 were run under native conditions on polyacrylamide gel electrophoresis. Both rgp140 proteins migrated to approximately 420 kDa, while rgp120 migrated to the expected molecular size of 120 kDa. (B) SIV/17E-CL and SIVmac239 trimeric rgp140 proteins were characterized for binding to sCD4 by ELISA. Both trimeric proteins bound sCD4 at levels comparable to those of native viral (SIV/17E-CL) envelope proteins.
blots and ELISAs (9, 45). In the current study, we measured the relative affinity of rhesus MAb binding to SIV recombinant gp140 envelope proteins by ELISA and included a comparison of reactivity to the homologous, neutralization-sensitive SIV/ 17E-CL envelope with reactivity to the neutralization-resistant SIVmac239 rgp140. For these studies, SIVmac239 or SIV/ 17E-CL rgp140 antigens were expressed using a vaccinia virus expression system (14). Secreted, purified envelope proteins were characterized on native polyacrylamide gels. Both SIVmac239 and SIV/17E-CL rgp140 antigens migrated predominantly as 420-kDa homogenous proteins (Fig. 2A), consistent with findings by Center and colleagues demonstrating that secreted forms of SIVmac239 rgp140 are trimeric (6, 7). While SIV/17E-CL rgp140 migrated slightly faster than SIVmac239 rgp140, we attributed this difference to amino acid changes between the two envelope proteins that result in SIV/ 17E-CL having two fewer potential N-linked glycosylation sites (1). To further demonstrate that these envelope trimers were functional, rgp140 proteins were assayed for their ability to bind soluble CD4 by ELISA. As shown in Fig. 2B, both SIV/
17E-CL and SIVmac239 rgp140 proteins bound sCD4 at levels comparable to those of native viral envelope proteins. Once characterized, SIV rgp140 envelope proteins were used to quantitatively measure rhesus MAb binding in a ConA ELISA as previously described (9, 45). The relative affinity of reference neutralizing and nonneutralizing MAbs was determined by calculating the half-maximal binding to SIV rgp140 proteins (Fig. 3). Differences in the quantitative reactivities of specific MAbs for one or both rgp140 antigens were observed; however, the relative affinities determined by ELISA were not different when reactivities of neutralizing and nonneutralizing MAbs with the target rgp140 protein were compared. For example, nonneutralizing MAbs 3.10A and 5.5B had the highest affinities measured against SIV/17E-CL rgp140 (2.4 ⫻ 10⫺10 and 1.1 ⫻ 10⫺10, respectively). In contrast, the neutralizing MAbs demonstrated similar affinities for SIV/17E-CL rgp140, and these affinities were comparable to those of the remaining nonneutralizing MAbs, ranging from 1.6 ⫻ 10⫺8 to 5.0 ⫻ 10⫺10. Finally, we observed relative MAb affinities for SIVmac239 that were similar to those observed for SIV/17E-
FIG. 3. Relative affinities of rhesus MAb binding to SIV rgp140 in a ConA ELISA. Levels of binding to SIV/17E-CL rgp140 (solid bars) did not distinguish between neutralizing and nonneutralizing MAbs. In addition, no quantitative differences in rhesus MAb reactivity to SIV/17E-CL or SIVmac239 (shaded bars) rgp140 were observed.
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TABLE 3. Comparison of MAb-SIVmac239 rgp140 binding kinetics at 25 °C and 37 °C 25 °C MAb ka (m
3.8E 3.11H 1.10A
⫺1
s
⫺1
4.7 ⫻ 10 3.9 ⫻ 104 1.9 ⫻ 105 4
)
kd (s
⫺1
37 °C ) ⫺5
6.4 ⫻ 10 5.9 ⫻ 10⫺5 4.2 ⫻ 10⫺4
KD (M)
ka (m
⫺9
1.4 ⫻ 10 1.5 ⫻ 10⫺9 2.1 ⫻ 10⫺9
CL. Thus, the relative affinities measured by ELISA also failed to reveal quantitative differences in reactivity to the neutralization-sensitive SIV/17E-CL envelope versus the neutralization-resistant SIVmac239 rgp140 envelope proteins. Effect of temperature on rhesus MAb binding as measured by SPR. Since MAb-envelope protein reactivity by ELISA failed to discriminate between neutralizing and nonneutralizing antibodies, we characterized the abilities of rhesus MAbs to bind SIV envelope proteins in “real time” by using SPR. Our first goal was to determine the optimum conditions for measuring the quantitative binding properties of rhesus MAbs to SIV envelope proteins. Previous studies have employed SPR to examine the interactions between HIV-1 gp120 and V3-specific monoclonal antibodies in order to demonstrate differences in MAb binding to gp120 that discriminate between neutralizing and nonneutralizing MAbs (48). In the current study, we analyzed a panel of rhesus MAbs that recognize epitopes spanning the entire SIV gp120 molecule. We utilized modified procedures taking into account advances in instrument sensitivity and parameters of experimental design that have increased the reliability of kinetic data. These technical advances, combined with the discovery that SIV rgp140s assume functional trimeric structures that presumably mimic envelope spikes on virions, allowed for kinetic measurements of 1:1 Langmuir binding interactions with trimeric envelope proteins (7). Previous studies analyzed antibody binding to HIV-1 envelope proteins at 25°C (48). Since interactions between virus and antibodies in vivo occur at 37°C, we analyzed rhesus MAb binding to SIVmac239 rgp140 at both temperatures using a Biacore 3000. For these studies we chose three rhesus MAbs from three distinct binding domains: 3.8E, a nonneutralizing MAb recognizing a conformational epitope in the N terminus of SIV gp120; 3.11H, a neutralizing MAb recognizing a linear V3 epitope; and 1.10A, a neutralizing MAb recognizing a conformational epitope in the C terminus of SIV gp120. The sensorgrams are shown in Fig. S1 in the supplemental material. Using Biaevaluation 4.1 software, we determined the kinetic rates and affinity constants for these three rhesus MAbs by global fitting to a 1:1 Langmuir model. As shown in Table 3, similar ka, kd, and KD of three rhesus MAbs for SIVmac239 rgp140 were observed at 25°C. Increasing the reaction temperature to 37°C did not alter the KD. However, increasing the temperature from 25°C to 37°C did increase the ka and kd of all three MAbs. For example, MAbs 1.10A and 3.11H exhibited kd at 37°C that were 1 to 2 log units higher (1.2 ⫻ 10⫺3 s⫺1 and 1.1 ⫻10⫺3 s⫺1, respectively) than the kd at 25°C (4.2 ⫻ 10⫺4 s⫺1and 5.9 ⫻ 10⫺5 s⫺1, respectively). Similar increases in ka at 37°C compared to 25°C were also observed (Table 3). Based on the observed effect of temperature on rate constants, we chose
⫺1
⫺1
s
3.2 ⫻ 10 2.6 ⫻ 105 4.9 ⫻ 105 4
)
kd (s⫺1) ⫺4
2.1 ⫻ 10 1.1 ⫻ 10⫺3 1.2 ⫻ 10⫺3
KD (M)
6.9 ⫻ 10⫺9 4.3 ⫻ 10⫺9 2.5 ⫻ 10⫺9
to perform the comprehensive comparisons of antibody binding to variant envelope proteins at the biologically relevant temperature of 37°C. Comparison of neutralizing and nonneutralizing MAb binding kinetics to the neutralization-sensitive SIV/17E-CL envelope protein by SPR. Our first goal was to determine if differences in binding to SIV envelope proteins could discriminate between neutralizing and nonneutralizing MAbs. The SIVmac239 molecular clone is one of the most widely characterized viruses used for mutagenesis and vaccine studies (43). However, neutralization of this virus in vitro is difficult to achieve (23). Previous studies demonstrated the ability of these rhesus MAbs to neutralize SIV/17E-CL but not SIVmac239 in vitro (9, 45). For this reason, we compared binding properties of neutralizing and nonneutralizing MAbs to a neutralizationsensitive envelope protein, SIV/17E-CL rgp140, in SPR assays conducted at 37°C (1). The sensorgrams from these studies are shown in Fig. S2 in the supplemental material. Kinetic rates of association and dissociation and affinity constants were determined by fitting a 1:1 Langmuir model. The ka ranged from 6.9 ⫻ 104 M⫺1 s⫺1 to 3.2 ⫻ 106 M⫺1 s⫺1 for neutralizing MAbs and were 1.1 ⫻ 105 M⫺1 s⫺1 for both nonneutralizing MAbs (Table 4), with similar median association rate constants for neutralizing and nonneutralizing MAbs (4.7 ⫻105 M⫺1 s⫺1 and 1.1 ⫻105 M⫺1 s⫺1, respectively) (Fig. 4A). Similarly, the kd for the two sets of MAbs also overlapped, ranging from 1.5 ⫻ 10⫺4 s⫺1 to 7.5 ⫻ 10⫺4 s⫺1 for neutralizing MAbs and from 3.7 ⫻ 10⫺4 s⫺1 to 4.0 ⫻ 10⫺4 s⫺1 for nonneutralizing MAbs (Table 4), with a median kd for neutralizing MAbs of 3.3 ⫻10⫺4 s⫺1 compared to 3.9 ⫻10⫺4 s⫺1 for nonneutralizing MAbs (Fig. 4B). Finally, KD demonstrated that all MAbs tested were of high affinity, i.e., they all bound SIV/17E-CL rgp140 in the nanomolar range or lower (Table 4; Fig. 4C). Taken together, the results of these SPR assays demonstrated no significant differences in kinetic rate or affinity constants between neutralizing MAbs and nonneutralizing MAbs. Comparison of rhesus MAb binding to neutralization-resistant SIVmac239 and neutralization-sensitive SIV/17E-CL rgp140 envelope proteins by SPR. To determine whether sensitivity to neutralization of SIV strains in vitro could be related to differences in MAb binding, we compared the properties of rhesus MAb binding to the neutralization-resistant SIVmac239 rgp140 with those of binding to the neutralization-sensitive SIV/17E-CL rgp140 by kinetic SPR analyses. In general, we observed marked differences in the rates of MAb binding between the two envelope proteins (Table 4; Fig. 5). The association rate for a specific rhesus MAb with SIVmac239 was as much as 1 log unit higher than that observed for SIV/17E-CL. For example, the ka of MAb 1.10A for SIVmac239 rgp140 was 5.7 ⫻ 105 M⫺1 s⫺1, while that for SIV/17E-CL was 7.5 ⫻ 104
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TABLE 4. Kinetics of MAb binding to recombinant gp140 envelope proteins at 37°C MAba
SIV 17E-CL 5
ka (10 ) (m
Nonneutralizing* 3.8E 3.10A 5.5B 3B3 Neutralizing 1.11A 4.7A 3.11E 3.2C E31 1.10A C26 3.11H a b
⫺1
⫺1
s
)
ka (10
⫺3
) (s
⫺1
SIVmac239 )
KD (10
⫺9
) (M)
DNBb 1.1 (⫾0.21) 1.1 (⫾0.10) DNB
DNB 0.40 (⫾0.02) 0.37 (⫾0.07) DNB
DNB 3.7 (⫾0.85) 2.1 (⫾0.33) DNB
5.9 (⫾1.4) 0.97 (⫾0.04) 5.8 (⫾0.22) 32 (⫾0.09) 3.5 (⫾0.01) 0.75 (⫾0.01) 10 (⫾0.63) 0.69 (⫾0.09)
0.26 (⫾0.17) 0.35 (⫾0.11) 0.36 (⫾0.01) 0.45 (⫾0.03) 0.30 (⫾0.05) 0.75 (⫾0.13) 0.15 (⫾0.02) 0.27 (⫾0.15)
0.43 (⫾0.29) 3.6 (⫾1.2) 0.63 (⫾0.04) 0.10 (⫾0.15) 0.85 (⫾0.13) 10 (⫾0.20) 0.14 (⫾0.01) 3.7 (⫾2.2)
5
ka (10 ) (m
⫺1
s
⫺1
0.53 (⫾0.12) 2.2 (⫾0.52) DNB 3.5 (⫾0.27) 26 (⫾12) 11 (⫾0.22) 33 (⫾10) 270 (⫾1.1) 17 (⫾0.1) 5.7 (⫾0.59) 62 (⫾1.7) 2.8 (⫾0.08)
)
kd (10⫺3) (s⫺1)
KD (10⫺9) (M)
0.48 (⫾0.13) 0.70 (⫾0.18) DNB 1.3 (⫾0.04)
9.0 (⫾0.48) 3.4 (⫾0.33) DNB 3.6 (⫾0.40)
0.85 (⫾0.16) 0.32 (⫾0.08) 0.92 (⫾0.09) 0.62 (⫾0.04) 1.8 (⫾0.02) 1.5 (⫾0.2) 1.4 (⫾0.07) 1.3 (⫾0.02)
0.38 (⫾0.23) 0.30 (⫾0.07) 0.30 (⫾0.12) 0.023 (⫾0.003) 1.1 (⫾0.2) 2.6 (⫾1.2) 0.22 (⫾0.01) 4.6 (⫾0.20)
Designations for neutralizing and nonneutralizing antibodies are based on data for SIV/17E-CL. DNB, does not bind by Biacore.
M⫺1 s⫺1 (Table 4). Similar differences were observed for all MAbs analyzed, both neutralizing and nonneutralizing. The median ka for MAb binding to SIVmac239 rgp140 was 2.6 ⫻ 106 M⫺1 s⫺1, compared to the median ka of 5.8 ⫻ 105 M⫺1 s⫺1 for MAb binding to SIV/17E-CL rgp140 (Fig. 5A). Rhesus MAb dissociation from SIVmac239 rgp140 was also faster than that observed for SIV/17E-CL rgp140. The kd for the MAb
FIG. 4. MAb-rgp140 binding interactions do not discriminate between neutralizing and nonneutralizing MAbs. Kinetics of neutralizing (■) and nonneutralizing (䊐) rhesus MAb binding to SIV/17E-CL identified no significant differences in the median ka (A), kd (B), or KD (C).
1.10A-SIVmac239 rgp140 interaction was 1.5 ⫻ 10⫺3 s⫺1, compared to 7.5 ⫻ 10⫺4 s⫺1 for SIV/17E-CL (Table 4). In addition, the median kd for SIVmac239 was approximately one-half log unit higher than the median kd observed for SIV/17E-CL (9.2 ⫻ 10⫺4 s⫺1 and 3.5 ⫻ 10⫺4 s⫺1, respectively) (Fig. 5B). These
FIG. 5. Rates of MAb-rgp140 binding discriminate between neutralization-sensitive and neutralization-resistant SIV. (A and B) The kinetics of rhesus MAb binding to SIV/17E-CL (■) and SIVmac239 (E) demonstrated higher ka (A) and kd (B) for SIVmac239 than for SIV/17E-CL; these differences were statistically significant (P ⫽ 0.0078 and 0.0156, respectively). (C) No difference in the median KD was observed.
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observed differences for the rates of association and dissociation were considered statistically significant (P ⫽ 0.0078 and P ⫽ 0.0156, respectively). Interestingly, despite marked differences between the kinetics of MAb binding to SIVmac239 rgp140 and that to SIV/17E-CL rgp140, the median KD calculated for MAb binding to the variant envelope proteins were similar (3.0 ⫻ 10⫺10 M and 6.3 ⫻ 10⫺10 M, respectively; P ⫽ 0.4609) (Fig. 5C). These results demonstrated for the first time that differences in antibody binding kinetics predict the ability of a particular MAb to neutralize variant SIVs in vitro. DISCUSSION We report for the first time that differences in the kinetic rates of antibody binding to recombinant envelope proteins by SPR predicted the neutralization sensitivity of the corresponding SIV strain. Despite similar KD, significant differences in rhesus MAb ka and kd discriminated between binding to the neutralization-sensitive SIV/17E-CL rgp140 and the neutralization-resistant SIVmac239 envelope proteins (Table 4; Fig. 5). The rates of MAb association with and dissociation from SIVmac239 were significantly higher than the kinetic rates observed for SIV/17E-CL. While increased rates of antibody association may be desirable, the faster dissociation of MAbs from SIVmac239 rgp140 results in a less-stable antibody-envelope interaction than that observed for antibody and SIV/ 17E-CL rgp140. This instability of MAb-rgp140 binding identifies one mechanism for the relative resistance of SIVmac239 to antibody-mediated neutralization. These differences in antibody binding between the two envelope proteins suggest that antibody recognition of the SIVmac239 envelope protein is fundamentally different from recognition of the neutralization-sensitive SIV/17E-CL envelope protein. SIVmac239 is a molecular clone of a pathogenic virus isolate that infects cells in a CD4-dependent manner and is highly resistant to antibody-mediated neutralization (23). In contrast, SIV/17E-CL is a recombinant molecular clone that differs from SIVmac239 by only 10 amino acids, 9 of which are located in or N-terminal to the V3 loop, and is capable of infecting target cells in a CD4-independent manner (1). We have demonstrated that the difference in neutralization sensitivity between the two virus strains cannot simply be explained by differences in envelope content in the virion (Fig. 1; Table 2). One explanation for the observed differences in antibody binding to the two envelope proteins is the potential for differences in MAb epitope availability. It has been proposed that neutralizing antibody epitopes on CD4-independent envelope proteins may be more accessible, since the envelope is suggested to be in a more “open” or “triggered” conformation (3, 17, 22, 42). In addition, increases in sensitivity to MAbmediated neutralization have also been reported for viruses containing limited amino acid changes with respect to SIVmac239 or containing deletions of glycosylation sites and/or variable regions that have previously been shown to limit antibody recognition (24). Despite differences in amino acid sequence between SIV/17E-CL and SIVmac239, to our surprise, we observed faster MAb association with SIVmac239 than with SIV/17E-CL rgp140. These data provided evidence that epitope availability alone cannot account for differences in neutralization sensitivity. In contrast, it is possible that the
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structure of SIVmac239 may allow for prominent visibility of at least a portion of the MAb epitope(s) but that an additional conformational change (i.e., CD4 binding) is required to fully expose the remainder of the epitope to allow for complete and more stable MAb binding. Alternatively, the significant difference in dissociation rates between the two envelope proteins, where less-stable antibody-envelope interactions were observed with the nonneutralizing SIVmac239 envelope protein, suggested that the stability of the antibody-envelope interaction (as determined by the dissociation rate) is more important in distinguishing between those viruses that will be neutralized and those that are resistant to neutralization by the same antibody. Since the 10-amino-acid differences that exist between SIV/17E-CL and SIVmac239 rgp140 proteins do not account for differences in epitope availability, it is likely that there are subtle yet important differences in envelope structure necessary for the stability of neutralizing antibody binding between the two envelope proteins. Studies to elucidate the binding epitopes of these conformationally dependent neutralizing MAbs are in progress and will provide important information about potential structural differences between the two proteins. In support of these conclusions, binding of HIV-1 gp120 with a CCR5 surrogate (17b) decreased the affinity of the sCD4-gp120 interaction by decreasing the on rate and increasing the off rate (55). Conversely, treating HIV-1 gp120 with sCD4 resulted in an increased affinity of gp120 for 17b, primarily by increasing the on rate (55). This observation can also be applied to the observations we have made for SIV/17E-CL and SIVmac239 and may allow us to explain the differences in neutralization sensitivity between the two viruses. For example, the lower off rates (higher binding stability) observed for SIV/17E-CL could result in enhanced neutralization by two mechanisms that could work independently or in combination: MAb binding could (i) sterically occlude the coreceptor binding site or (ii) decrease the affinity of the SIV/17E-CL envelope for CD4 such that the MAb competes with CD4 for envelope binding. While we have shown previously by ELISA that these MAbs do not directly inhibit CD4 binding (9), further analyses by Biacore may demonstrate MAb interference related to the stability of the CD4-envelope interaction. In addition, Zhang et al. also showed that CD4 binding increased the affinity of gp120 for a coreceptor surrogate (17b) by increasing the on rate (55). The fact that the MAbs exhibit higher off rates for SIVmac239 may allow an opportunity for CD4 to bind when the MAb dissociates, allowing it then to quickly recruit and bind coreceptor and gain entry into the cell even in the presence of antibody. Once again, the higher dissociation rates appear to be more important in defining a possible mechanism for the relative resistance of SIVmac239 to neutralization, since MAbs are simply not remaining bound to the SIVmac239 envelope long enough to inhibit or destabilize CD4 binding. Additional studies to evaluate the rates of CD4 and coreceptor binding in the presence of these MAbs may provide insight into this mechanism. These results may lend further insight into the increased sensitivity to neutralization observed with CD4-independent viruses. CD4-independent strains of both SIV and HIV-1 are easier to neutralize than CD4-dependent strains (17, 28, 42, 54), and this sensitivity to neutralization is due, in part, to an
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FIG. 6. Lack of correlation between rhesus MAb affinities determined by SPR and ConA ELISA analyses. Rhesus MAb affinities for SIV/17E-CL rgp140 (■) and SIVmac239 rgp140 (E) were determined by SPR (Biacore) and half-maximal binding in a ConA ELISA. As indicated by the regression analysis r2 values (solid line, SIV/17E-CL; dashed line, SIVmac239), there was no correlation between affinities determined by SPR and those obtained in a solid-phase ELISA.
altered conformation of CD4-independent envelope proteins that results in the relative exposure of the coreceptor binding site (3, 17, 22, 42). Based on this combination of decreased CD4 dependence and increased sensitivity to antibody-mediated neutralization, it has been suggested that one mechanism of neutralization may involve inhibition of interactions necessary for efficient virus binding and entry via specific cell surface receptors (13, 15, 42). In the current study, we observed significant differences in the rates of MAb binding that demonstrated a more stable antibody interaction with the neutralization-sensitive, CD4-independent SIV/17E-CL envelope protein than with the CD4-dependent SIVmac239 rgp140. Thus, we propose that the increased stability of the antibodySIV/17E-CL rgp140 interaction may provide one mechanism for the increased sensitivity to neutralization exhibited by CD4-independent viruses in vitro. It will be of interest to extend these studies to additional envelope proteins, especially those exhibiting intermediate sensitivity to neutralization and CD4 dependence. Results from these kinetic analyses were in contrast to the measurement of relative affinities by ELISA, where differences in rhesus MAb binding to SIVmac239 and SIV/17E-CL rgp140 proteins failed to identify differences between the two envelope proteins (Fig. 3). The lack of correlation between measurements of MAb affinity by SPR and ELISA was confirmed by regression analyses (r2 ⫽ 0.004 for SIVmac239; r2 ⫽ 0.087 for SIV/17E-CL) (Fig. 6), clearly demonstrating the inability of solid-phase ELISA to determine solution-phase affinities as measured by SPR. While this finding is not novel, many studies continue to report antibody affinity measurements determined by ELISA. These results highlight the need to use appropriate and sensitive techniques to evaluate properties of antibody binding (12). Finally, we observed that rhesus MAb binding properties (association, dissociation, and affinity) for SIV/17E-CL rgp140 did not discriminate between neutralizing and nonneutralizing antibodies (Table 4; Fig. 4). These results demonstrated that antibody binding properties alone were not sufficient to predict neutralization. These data are in contrast to previous studies by VanCott et al. demonstrating that rates of dissociation from
J. VIROL.
HIV-1 gp120 proteins correlate with the neutralization abilities of a panel of V3-specific MAbs (48). Two potential explanations for the differences observed in the current study exist. First, it is likely that the measurement of MAb binding to trimeric envelope proteins will result in kinetic binding properties different from those found in studies using monomeric gp120. Studies to compare the kinetic binding properties between gp120, rgp140 trimers, and native viral envelope proteins are in progress. Second, our study demonstrated that differences in the rates of antibody binding were not sufficient to predict neutralization of diverse envelope determinants. Further studies analyzing binding properties of MAbs directed to single epitopes (e.g., V3) that differ in their abilities to neutralize virus may support previous studies with HIV-1 gp120 and provide correlations between MAb binding properties and neutralization. At present two predominant models for neutralization of SIV and HIV-1 exist. The first model, also referred to as the “occupancy model,” proposes that neutralization sensitivity is related to the density of an antibody and its high-affinity interaction with envelope spikes on the virion, independent of the epitope, requiring a threshold level of antibody binding to limit infectivity (27, 38–40). Alternatively, neutralization of virus by antibody has been associated with differences in qualitative antibody binding properties, i.e., antibody affinity and epitope specificity (9, 15, 26, 56). Our data provide more support for the latter model of antibody-mediated virus neutralization. We provide evidence that epitope specificity is a component of neutralization, since, first, all MAbs bound SIV/17E-CL with high affinity yet only those MAbs that clustered in the Cterminal half of gp120 neutralized SIV/17E-CL. In contrast, those MAbs that recognized epitopes in the N terminus of gp120, including the V1 and V2 regions, or in the very C terminus of gp120 failed to neutralize SIV/17E-CL, despite binding with an affinity similar to that of the neutralizing MAbs (9, 45). Second, MAb binding to SIV/17E-CL and SIVmac239 demonstrated significant differences in the rates of binding to the two envelope proteins but not in the affinity of the binding. Importantly, the difference in the dissociation rate indicated that the binding to SIV/17E-CL is more stable than the binding to SIVmac239. This suggests that more antibody remains bound to the SIV/17E-CL virion, occupying more envelope trimers, than the same MAb does on the SIVmac239 virion over time. Taken together, the results presented in this study and others support the hypothesis that neutralization involves a combination of epitope specificity and binding properties (i.e., rates and affinity) (9, 24, 56). Evidence from extensive animal and recent human vaccine trials strongly indicates that an effective HIV-1 vaccine will need to induce both broadly reactive neutralizing antibodies and cell-mediated immunity. Potent and broadly reactive antibodies have been identified in sera from HIV-1-infected individuals, demonstrating that elicitation of protective antibodies as part of the B-cell repertoire is possible (4, 56). However, inducing these broadly neutralizing antibodies with candidate vaccine strategies in both the monkey model and human vaccine trials has been difficult. A major limitation has been the lack of knowledge about what constitutes a protective neutralizing antibody response. Results from this study provide novel insight into one mechanism of neutralization, demonstrating
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fundamental differences in the antibody-envelope interactions between neutralization-sensitive and neutralization-resistant viruses. Further, these studies indicate that the kinetic rates of antibody binding to recombinant SIV envelope proteins correlate with the neutralization sensitivities of the corresponding virus strains. It is likely that both kinetic properties of antibody binding, including unidentified properties of envelope proteins involved in the stability of antibody binding, and epitope specificity are necessary components of a potent neutralizing antibody response. Studies to define additional mechanisms of antibody-mediated neutralization and to develop assays that effectively discriminate between neutralizing and nonneutralizing antibodies will be necessary for the identification of immunogens capable of achieving protection against HIV-1 infection.
13. 14.
15.
16.
17.
ACKNOWLEDGMENTS
18.
We thank Richard Day for guidance with the statistical analyses and Ted Ross and Donald Sodora for helpful discussions. This work was supported by NIH grants AI 28243 and AI 52058.
19.
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