Targeting carbohydrate antigens in HIV vaccine development

Vaccine 23 (2005) 2168–2175

Targeting carbohydrate antigens in HIV vaccine development Anastas Pashov a , Gabriela Canziani b , Stewart MacLeod a , Jason Plaxco a , Behjatolah Monzavi-Karbassi a , Thomas Kieber-Emmons a, ∗ a

Department of Pathology, University of Arkansas for Medical Sciences, ACRC #824, UAMS, 4301 Markham Street, Little Rock, AR 72205, USA b Center for Biomolecular Interaction Analysis, The University of Utah, Salt Lake City, UT, USA Available online 15 February 2005

Abstract Peptide mimotopes provide a strategy to augment human immunodeficiency virus 1 (HIV-1) specific carbohydrate reactive immune responses. Their antigenic and immunological properties will depend on the optimization of motif clustering and multimerization. We observe that structural variants of the same mimetic motif, linear versus cyclic, can be used to tune the properties of the antibodies elicited. The expansion of the database of mimotope sequence motifs can be increased by analyzing structures that bind to HIV directed monoclonal antibody 2G12 and the lectin Concanavalin A (Con A), fostering new mimotope designs. Such analysis indicates that these reagents bind to subsets of mannosyl antigens on the envelope (env) protein. © 2005 Elsevier Ltd. All rights reserved. Keywords: HIV-1; Concanavalin A; Carbohydrate; Mimotope; Multiple antigen mimotope; MAM

1. Introduction Targeting carbohydrate antigens expressed on the envelope (env) protein of human immunodeficiency virus 1 (HIV1) has become of interest with the realization that the human antibody 2G12 which neutralizes a broad range of HIV-1 isolates and is in clinical trials [1], binds an unusually dense cluster of mannosyl glycans on the “silent” face of the gp120 env [2–6]. Early observations that lectins and carbohydrate reactive antibodies could affect some level of virus neutralization highlighted the biological significance of env expressed carbohydrate antigens [7–9]. Development of antibodies that block the transmission of the virus may also be considered as the C-type lectin dendrtic cell (DC)-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-Sign) plays a role in virus infection and transmission [10]. Adhesion is not solely mediated by DC-Sign as fucosylated lactosamines expressed on the env protein and on HIV infected cells also play a role in adhesion [11]. Over the last several years, our laboratory has conceptualized and implemented a strategy to augment carbohydrate ∗

Corresponding author.

0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.01.045

reactive immune responses making use of peptide mimotopes of carbohydrate antigens to facilitate the T-cell dependent induction of carbohydrate reactive antibodies and activation of T-cells. Mimotopes can induce serum antibodies in mice that can bind to the env protein and neutralize Lab isolates in cell free infection assays [12,13], and can facilitate the induction of carbohydrate reactive antibodies to the env protein in prime-boost strategies [14]. To foster new mimotope designs we further expand the repertoire of mimotope structures of HIV associated carbohydrate antigens by considering structural variants and describe new peptides reactive with the antibody 2G12 and the lectin Concanavalin A (Con A),

2. Materials and methods 2.1. Immunization Several different vaccine formats that include peptide mimotope encoded DNA and peptides synthesized as multiple antigen peptides (MAPs) were tested. For DNA immunization, a respective peptide sequence was converted

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to its DNA counterpart and inserted into the pcDNA3 (Invitrogen, San Diego, CA) vector, as described earlier [15]. Peptides synthesized as MAPs were of 8 mere or 4 mere forms (Bio-Synthesis, Inc., Lewisville, TX). Sixto eight-week-old BALB/c female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Immunization protocols and procedures followed our previous studies [13–15].

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Table 1 Comparative thermodynamics of binding of ligands to Con A by isothermal titration calorimetry Reactive ligand

Ka (M−1 )

G (kcal mol−1 )

912 ␣-d Methylmannopyranoside Trimannoside Pentasaccharide 911

1.1 × 104

5.6 5.3 7.8 8.1 7.4

0.82 × 104 49 × 104 92 × 104 26 × 104

2.2. ELISA HIV-1 IIIB gp120 was used to assess reactivity of collected serum by standard ELISA as described previously [12]. The specificity of the phage selected was tested in ELISA using the detection module kit of RPAS (27-9402-01, Amersham) according to the manufacturer’s instruction. For the inhibition assay, the phage suspensions were coated on the plate and incubated with 2G12 at 0.2 ␮g/ml in the presence of different sugars as inhibitors. This concentration of 2G12 was previously determined to yield sub half-maximal binding to gp160 in an equivalent experimental setting. The binding was visualized using alkaline phosphatase conjugated anti-human IgG (A3312, Sigma). To test the binding of 2G12, human IgG, Con A and Jacalin to peptides, plates were coated with the peptides (1 ␮g/ml). 2G12 or biotinylated lectins were added and binding visualized using alkaline phosphataseconjugated anti-human IgG or Streptavidin (Sigma), respectively. 2.3. Biosensor binding experiments MAP mimetics were coupled to the sensor surface of B1 sensor chip (Biacore, Uppsala, Sewden). Binding of Con A (Sigma) was measured using a Bia3000 (Biacore, Uppsala, Sewden) biosensor, at 25 ◦ C. Non-specific interaction with the matrix was diminished using 10 mM phosphate buffered, 0.005% polysorbate-20 solution, pH 7.0 with 500 mM NaCl. The flow was kept at 50 ␮l/min. Binding interactions were measured without added bivalent cations,

nor EDTA or EGTA. The sensor surface was regenerated using duplicate 5–10 ␮l pulses of 1 M NaCl, 5 mM NaOH (pH ∼10–11) at 100 ␮l/min. Binding was measured in triplicate samples of Con A at concentrations 14.375–460 nM per experiment in two-fold dilution series. Single exponential dissociation-rate fits of the direct binding data were performed using BIA Evaluation 3.1 and Microsoft Excell 4.0 Macros. 2.4. Flow cytometry Transfected cell lines were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Chinese hamster ovary cells (CHO)-PI and EE were obtained from Drs. Carol Weiss and Judith White. The HXB2 Env gene was introduced to CHO-PI cells using the construct pEE14-envelope that also contains a glutamine synthetase minigene for selection purpose. Cells stably transfected with the expression vector pEE14, which does not contain the gene for the Env protein, and referred to as CHOEE cells were used as controls. Fluorescence rgp120 HIV-1 IIIB was purchased from ImmunoDiagnostic, Inc. (Woburn, MA). Cell binding and competition study was performed as reported [11]. 2.5. Generation of DCs and cell adhesion assay Human DCs were provided as a part of LGMTM -3 BulletKit® by Clonetics (BioWhittaker, Inc., Walkersville, MD), where cells were plated and developed in vitro for 6

Fig. 1. Binding of Con A to MAP911 and MAPD002 measured by SPR. At low Con A concentration (0.02–0.12 mM), Con A bound MAP911 to form a complex of higher stability (A, thin line), kd = 1.7 × 10−3 ± 6.3 × 10−5 s−1 , compared to that of Con A bound to MAPD002 (panel A, thick line), kd = 4.9 × 10−3 ± 1.4 × 10−5 s−1 . No significant difference was observed, however, in the stability of each complex at high concentration of Con A (0.26–1.12 mM) (panel B).

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Fig. 2. (A) Serum from MAP911-immunized mice inhibits adhesion of dendritic cells to A1953 cells. A1953 cells were washed, labeled with Calcein AM (molecular probes) and added to wells containing confluent DCs in the presence of medium (DMEM, 1% FBS) only or medium supplemented with 1:40 dilution of serum from MAP911 immunized and na¨ıve mice. Percentage adhesion was calculated as following: {(experimental-background)/(maximumbackground)} × 100. The asterisk stands for significant difference from the percentage of adhesion from the control (Student’s t-test, p < 0.05). (B) Inhibition of DC adhesion to A1953 cells with serum (1:40) from mice using different prime and boost regimes. Assay was performed as in A.

Fig. 3. (A) Binding of MAPD002 serum (1:40) to CHO cells transfected with gp120 (CHO-PI) or with an empty vector (CHO-EE) compared to preimmune serum measured by flow cytometry. The thin line histogram represents the binding of preimmune serum to the cells. The numbers above the histograms represent the difference in median fluorescence intensity (MFI). The gp120 transfected cell line expresses almost twice as much MAPD002 related epitopes than the one transfected with an empty vector. (B). Fluorescent conjugated gp120 binds to human DCs and serum (1:40) collected from MAP911 or MAPD002 mice inhibits gp120 binding as measured by flow cytometry. Bars are S.D. estimates of three replications. The asterisk stands for significant difference from MFI of gp120 binding to DC (Student’s t-test, p < 0.05).

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Fig. 4. Peptides, selected for binding to monoclonal antibody 2G12 or 2G12 and Con A, containing consensus sequences. Peptides A, D and H were selected after adsorbing on BSA and three rounds of panning on 2G12. Peptides 1–5, 8–12, 14, 15, 17, 18 were selected after adsorbing the library selected in the previous step on human IgG and panning once more on 2G12 in the presence of human IgG (rounds 4 and 5). Peptides C1, C2, C4, C6, C8, C10, C11 and C12 were selected after the library from round 5 was panned on Con A and the bound phages eluted by ␣-d-methylmannopyranoside.

days as instructed by the provider using their recommended medium and cytokines. The cells adhered relatively firmly to the surface. The adhesion assay was done using VybrantTM Cell Adhesion Assay Kit (Molecular Probes, Inc., Eugene, OR), based on the manufacturer instructions, as described before [11].

the target. The procedure was the same as the first round of panning on 2G12, except that ␣-d-methyl mannopyranoside was used to elute the bound phage. At selected steps approximately 20 clones were sequenced on an Applied Biosystems Model 3100 Genetic Analyzer (Foster City, CA) using the sequencing primer provided with the kit.

2.6. Peptide library screening

2.7. Statistics

Peptides were selected using the Ph. D.-12 phage display library kit from New England Biolabs (Beverly, MA, cat. # E8110S) containing a random 12-mer peptide library. For the first three rounds of panning, the library was preadsorbed on BSA and panned on 2G12. The selected phage was amplified and (1–2) × 1011 of amplified phage used as input in the next round of panning. All subsequent rounds of panning were performed in the same manner, except the Tween 20 concentration in the wash solution was raised to 0.5%. The amplified eluent from the third round of panning was adsorbed on human Immunoglobulin G (huIgG) (Sigma–Aldrich, St. Louis, MO; catalog # I-4506). The non-binding phage was used as input for a fifth round of panning on 2G12 carried out in the presence of huIgG in the liquid phase. A sixth round of panning was performed with this eluent using Con A as

The mean fluorescent intensity in inhibition of binding experiments and the percentage of adherent cells in the inhibition of adhesion experiments were compared using the Student’s t-test. The level of significance was p < 0.05. The binding curves were compared using GLZ (generalized nonlinear models) module of Statistica (StatSoft).

3. Results 3.1. Antigenic properties of peptide mimotopes of HIV associated carbohydrate antigens Peptides representing a generalized (Trp)Tyr/X/Tyr (where X is a number of different residues) motif are sug-

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Fig. 5. Binding of representative phages from round three of the selection to 2G12 (filled diamonds) and human IgG (empty squares) in phage ELISA. Phages A, D, H represent the specific binding while the nonspecific phage B was considered non-binding since it did not bind more than the negative control (mixture of irrelevant phage) which itself showed significant binding of 2G12 (GLZ, p < 0.001). The comparison between the 2G12 and the human IgG coating demonstrates that the plates were coated with equivalent quantities of IgG.

gested to mimic elements of Core 1 and Core 2 structures shared among otherwise dissimilar carbohydrate structures [12]. As these peptides can bind to multiple carbohydrate binding proteins and can elicit antibodies that are polyspecific, we define these mimotopes as multiple antigen mimotopes (MAMs). Peptides with this motif are identified from random peptide libraries displayed on phage with reactivity to Con A [16,17]. Using pharmacophore design principles we showed that a peptide with the sequence YRYRYGRYRSGSYRYRYGRYRSGS, referred to as peptide 911, and its monomeric form YRYRYGRYRSGS referred to as peptide 912 were highly reactive with Con A [13]. These peptides display a free energy of association comparable to those reported for core trimannoside-Con A and pentasaccharideCon A interactions (Table 1) [13]. The isothermal titration calorimetry (ITC) analysis and its comparison with the data of multivalent sugars, point to certain fundamental differences in the overall binding mechanism. The differences between the multivalent binding systems are primarily attributed to the structural dissimilarities of the peptide and carbohydrate ligands, a conclusion indicated from molecular modeling studies [13] and crystallographic analysis [18]. As the generation of functional antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native antigen, we redesigned the putative RYRY motif of peptide 911 as a cyclic peptide. This peptide (D002) has the sequence RGGLCY-

CRYRYCVCVGR, which forms disulphide bridges between the 1st and 4th cysteines and between the second and third cysteines. The peptide 911 was made as a MAP on an 8lysine core while D002 was made as a MAP on 4-lysine core. Using surface plasmon resonance we observed that binding of Con A tetrameres to the MAPs is mechanistically complex (Fig. 1). The dissociation rate parameters for Con A interactions at high and low concentration with the peptide mimetics differ. Con A complexes at a MAP911 surface at low concentrations are more stable than complexes formed at a MAPD002 surface at comparable immobilization levels for the two mimetics. The multimeric nature of the ligands though precludes further study of the influence of peptide mimetic surface density on the avidity interaction of Con A. 3.2. Functional properties of serum antibodies to mimotopes We have utilized several vaccine formats for the induction of carbohydrate cross-reactive serum antibodies. These include MAPs [13], conjugates [12] and peptide mimotope encoded DNA plasmids [15]. We have recently examined the extent to which serum antibodies induced by peptide mimotopes can block the adhesion of gp120 and virus infected cells to human dendritic cells [11]. In this context adhesion of human DCs to HIV-infected cells is inhibited by serum antibodies induced by peptide 911 (Fig. 2A). Us-

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3.3. Development of new mimotopes to target carbohydrates on HIV envelope

Fig. 6. Inhibition of binding of phage expressing peptides (Ph) 4, 11 and 14 from the 5th round of selection as well as of gp160 to 2G12 by ␣-d methylmannopyranoside, mannan, fructose, mannose and glucose represented as the inverse of the IC50 as determined from the inhibition curves. Phages were coated on the plate at 1 ␮g/ml protein determined spectrophotometrically and HIV Env protein - at 0.2 ␮g/ml. 2G12 at 0.2 ␮g/ml was incubated with inhibitors in concentrations from 1 M (0.05 M for mannan) down to 10 ␮M.

ing DNA immunization and a combination of DNA, peptide and conjugated peptide in prime/boost settings [14] suggest that priming with 911 mimotope encoded DNA followed by boosting with peptide effectively generates serum antibodies that blocked the adhesion of infected cells to DCs (Fig. 2B). We also observe that the cyclic MAPD002 peptide can induce serum antibodies that bind specifically to gp120 expressed on CHO cells (Fig. 3A) and inhibits gp120 binding to human DCs as effectively as peptide MAP911 (Fig. 3B). These data imply that the generated serum to the linear MAP911 or MAPD002 competes with DC-SIGN in binding to gp120.

To expand our array of peptide mimetics to carbohydrate antigens of gp120, we have recently developed peptides using 2G12 as an isolating template (Fig. 4). A 12-mer phage display library was screened for peptides binding specifically to 2G12 by 3 rounds of panning (step 1), adsorption on human IgG and another round of panning on 2G12 in the presence of excess of human IgG (step 2) and panning on Con A with elution by high concentration of ␣-d-methylmannopyranoside (step 3). At each step a set of 20 peptides was randomly selected and analyzed. Peptides A, D and H from step 1, peptides 1–5, 8–12, 14, 15, 17, 18 from step 2 and peptides C1, C2, C4, C6, C8, C10, C11 and C12 from step 3 were selected for further studies because they belonged to one of 7 groups of peptides containing sequence consensus (Fig. 4). In ELISA the phage bound to 2G12 but not to human IgG (Fig. 5). The participation of carbohydrate binding sites in the peptide binding was demonstrated by inhibition of the binding of 2G12 to phages or HIV gp160 by mannose, fructose, glucose, mannan and ␣-d-methylmannopyranoside (Fig. 6). The binding was specifically inhibited by the mannose containing carbohydrates as well as by fructose but not by glucose. Three of the most prominent consensus bearing sequences were synthesized in MAP format. While Con A bound to all three, 2G12 did not bind to any of them (Fig. 7). Since the phage expressing the peptides did bind specifically to 2G12 it is possible that the binding is dependent on the way the peptide is clustered or on conformation induced by the pIII

Fig. 7. Binding of Con A, jacalin, 2G12 and human IgG to MAP forms of peptides from the last round of selection. “C consensus” indicates the sequence found in 6 of the peptides analyzed from that round. MAPs were coated on plates and incubated with biotinylated Con A, biotinylated Jacalin or 2G12.

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protein of the phage. The significant binding of a non-selected phage library to 2G12 indicates a possible interaction with other components of the phage particles. This could lead to a complex interaction facilitating the binding to low affinity peptide ligands. Interestingly two of the MAPs also bound the Jacalin lectin, which has different carbohydrate specificity from Con A.

4. Discussion Efforts directed toward developing HIV associated carbohydrate antigens as conjugated immunogens are underway [5,19,20]. The large number of potential carbohydrate targets makes the development of a multivalent carbohydrateconjugate vaccine far more complicated. Defining minimal “protective” epitopes on HIV associated carbohydrate antigens is therefore a first step. Alternatively, peptides that display conformations that induce antibodies reactive with a whole class of carbohydrate epitopes might be a way to simplify the approach to multivalent vaccine design. Immunization with a peptide or limited set of peptide immunogens programmed with this feature would elicit responses against a broad spectrum of carbohydrate epitopes expressed on the env protein [12]. In the development of mimotopes associated with mannose glycans we have used the mannose/glucose specific lectin Con A as a template receptor as Con A and 2G12 as they are anticipated to bind structural equivalents of Man9GlcNAc. The unique array of three binding sites on 2G12 for oligomannose is reminiscent of lectin binding, but their close proximity and the overall rigidity of the structure might define an array that provides specificity rather than avidity. We observe that the multivalent linear peptide binds to Con A as a multivalent cluster while the cyclic variant seems to bind as a monovalent form. Neither synthetic peptide binds to 2G12 nor do synthetic peptides identified on 2G12 in the present protocol bind back to 2G12. Thus, 2G12 and Con A might represent a model of two types of carbohydrate ligand presentation that are HIV-1 associated. Optimal design of carbohydrate mimotopes can be oriented to development of high affinity high specificity T cell dependent antibodies or, conversely, towards more broadly reactive lectin like antibodies that might depend on avidity for efficient binding. The former are considered the true aim of the technology while evolution has favored the second solution in the form of preimmune antibodies and IgM.

Acknowledgments We thank Dr. James Hoxie for the A1953 cells, Dr. H. Katinger for 2G12, and Drs. Carol Weiss and Judith White for the (CHO)-PI and -EE cells. This work was supported by NIH grant AI049092 to TKE.

References [1] Armbruster C, Stiegler GM, Vcelar BA, et al. A phase I trial with two human monoclonal antibodies (hMAb 2F5, 2G12) against HIV1. AIDS 2002;16(2):227–33. [2] Calarese DA, Scanlan CN, Zwick MB, et al. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 2003;300(5628):2065–71. [3] Scanlan CN, Pantophlet R, Wormald MR, et al. The carbohydrate epitope of the neutralizing anti-HIV-1 antibody 2G12. Adv Exp Med Biol 2003;535:205–18. [4] Wang LX, Ni J, Singh S, Li H. Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12: implications for HIV-1 vaccine design. Chem Biol 2004;11(1):127–34. [5] Lee HK, Scanlan CN, Huang CY, et al. Reactivity-based one-pot synthesis of oligomannoses: defining antigens recognized by 2G12, a broadly neutralizing anti-HIV-1 antibody. Angew Chem Int Ed Engl 2004;43(8):1000–3. [6] Sanders RW, Venturi M, Schiffner L, et al. The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J Virol 2002;76(14):7293– 305. [7] Hansen JE, Nielsen CM, Nielsen C, Heegaard P, Mathiesen LR, Nielsen JO. Correlation between carbohydrate structures on the envelope glycoprotein gp120 of HIV-1 and HIV-2 and syncytium inhibition with lectins. AIDS 1989;3(10):635–41. [8] Hansen JE, Clausen H, Nielsen C, et al. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization. J Virol 1990;64(6):2833–40. [9] Ezekowitz RA, Kuhlman M, Groopman JE, Byrn RA. A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus. J Exp Med 1989;169(1): 185–96. [10] Geijtenbeek TB, van Vliet SJ, van Duijnhoven GC, Figdor CG, van Kooyk Y. DC-SIGN, a dentritic cell-specific HIV-1 receptor present in placenta that infects T cells in trans-a review. Placenta 2001;22(Suppl. A):S19–23. [11] Monzavi-Karbassi B, Luo P, Cunto-Amesty G, et al. Fucosylated lactosamines participate in adhesion of HIV-1 envelope glycoprotein to dendritic cells. Arch Virol 2004;149(1):75–91. Epub 2003 Sep 2019. [12] Agadjanyan M, Luo P, Westerink MA, et al. Peptide mimicry of carbohydrate epitopes on human immunodeficiency virus [see comments]. Nat Biotechnol 1997;15(6):547–51. [13] Cunto-Amesty G, Dam TK, Luo P, et al. Directing the immune response to carbohydrate antigens. J Biol Chem 2001;30:30. [14] Monzavi-Karbassi B, Shamloo S, Kieber-Emmons M, et al. Priming characteristics of peptide mimotopes of carbohydrate antigens. Vaccine 2003;21(7-8):753–60. [15] Kieber-Emmons T, Monzavi-Karbassi B, Wang B, Luo P, Weiner DB. Cutting edge: DNA Immunization with minigenes of carbohydrate mimotopes induce functional anti-carbohydrate antibody response. J Immunol 2000;165(2):623–7. [16] Oldenburg KR, Loganathan D, Goldstein IJ, Schultz PG, Gallop MA. Peptide ligands for a sugar-binding protein isolated from a random peptide library. Proc Natl Acad Sci USA 1992;89(12): 5393–7. [17] Scott JK, Loganathan D, Easley RB, Gong X, Goldstein IJ. A family of concanavalin A-binding peptides from a hexapeptide epitope library. Proc Natl Acad Sci USA 1992;89(12):5398– 402. [18] Jain D, Kaur KJ, Goel M, Salunke DM. Structural basis of functional mimicry between carbohydrate and peptide ligands of con A. Biochem Biophys Res Commun 2000;272(3):843–9.

A. Pashov et al. / Vaccine 23 (2005) 2168–2175 [19] Li H, Wang LX. Design and synthesis of a template-assembled oligomannose cluster as an epitope mimic for human HIV-neutralizing antibody 2G12. Org Biomol Chem 2004;2(4):483–8. Epub 2004 Jan 2022.

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[20] Dudkin VY, Orlova M, Geng X, Mandal M, Olson WC, Danishefsky SJ. Toward fully synthetic carbohydrate-based HIV antigen design: on the critical role of bivalency. J Am Chem Soc 2004;126(31):9560–2.