1Department of Immunology, 3Department of Molecular Biology and 4Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
2 To whom correspondence should be addressed. E-mail: burton{at}scripps.edu; rpanto{at}scripps.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: AIDS vaccine/antigen engineering/gp120/HIV-1/neutralizing antibodies
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Design of an effective antigen that induces a neutralizing antibody response has been hampered by the fact that the virus has devised a number of ways to minimize antibody recognition of its surface envelope spikes, the major targets for any neutralizing antibody response. For example, antibodies are apparently largely incapable of accessing the gp41 transmembrane domain prior to binding of virus to target cells (Sattentau et al., 1995, 1999
), presumably owing to masking by the bulky gp120 surface domain, which partially caps gp41. Adjacent gp41 protomers and the close proximity of gp41 to the viral membrane most likely also limit access for antibodyantigen recognition. Gp120 is itself extensively covered by a dense array of host-derived carbohydrates, which render the underlying protein surfaces largely non-immunogenic. Furthermore, a number of highly variable loops serve to direct immune responses away from conserved epitopes within the CD4-binding site (CD4bs) (Kwong et al., 1998
, 2000
; Wyatt and Sodroski, 1998
; Wyatt et al., 1998
). Another potential target for antibodies, the conserved coreceptor-binding site (Rizzuto et al., 1998
; Rizzuto and Sodroski, 2000
), is partially masked by variable loops prior to CD4 binding (Wu et al., 1996
). The coreceptor binding site does become transiently exposed after conformational changes initiated by binding of gp120 to CD4 (Wu et al., 1996
; Xiang et al., 2002
), but it appears that spatial constraints resulting from binding of the virus to the target cell sterically hinder access of intact immunoglobulin molecules to epitopes within this region (Labrijn et al., 2003
).
Despite the plethora of viral defense mechanisms, a few broadly neutralizing monoclonal antibodies (mAbs) have been isolated that recognize conserved epitopes on gp41 and gp120. The two most potent, broadly neutralizing antibodies to gp120 are 2G12 and b12 (Burton et al., 2004). MAb 2G12 recognizes a conserved cluster of terminal mannose residues on the carbohydrate-covered face of gp120 (Trkola et al., 1996
; Sanders et al., 2002
; Scanlan et al., 2002
). Interestingly, this antibody has a highly unusual domain-exchanged structure (Calarese et al., 2003
), which enables it to recognize its multivalent carbohydrate epitope with high specificity and affinity (Calarese et al., 2003
). This dense array of oligomannoses, which has not been found as such on any other host cell or host glycoprotein, may, therefore, be an exploitable target for vaccine design (Calarese et al., 2003
; Lee et al., 2004
; Li and Wang, 2004
). The second mAb, b12, recognizes a conserved epitope that overlaps the CD4bs on gp120 (Burton et al., 1991
; Roben et al., 1994
). Because of its potency and broad neutralizing properties (Burton et al., 1994
; D'Souza et al., 1997
; Parren et al., 2001
; Veazey et al., 2003
), this antibody is considered a promising template for the design of an AIDS vaccine component aimed at the induction of highly cross-reactive antibodies with equivalent neutralizing properties (Poignard et al., 2001
; Saphire et al., 2001
; Burton, 2002
).
In this study, we focused primarily on mAb b12. To gain insight into the molecular requirements for b12 binding and, by inference, on how to design an antigen that specifically induces broadly neutralizing anti-CD4bs antibodies, gp120 was previously subjected to extensive alanine-scanning mutagenesis to identify residues that are critical for antibody binding (Pantophlet et al., 2003a). Four residues (G473, D474, M475 and R476) on the outer edge of the so-called Phe43 cavity (Kwong et al., 1998
; Wyatt et al., 1998
) were identified that, when replaced with alanine, abrogated or reduced binding of several non-neutralizing CD4bs mAbs (Pantophlet et al., 2003a
), whereas b12 binding was not affected. However, these alanine substitutions apparently did not affect binding of non- or weakly neutralizing antibodies to other gp120 epitopes. To inhibit binding of these antibodies, we adopted a strategy that could potentially dampen antibody responses in a selective manner (Garrity et al., 1997
). We have dubbed this approach immunofocusing (Pantophlet and Burton, 2003
). Consensus sequences for an N-glycosylation site with high probability of glycan incorporation (Gavel and von Heijne, 1990
; Kasturi et al., 1997
), Asn-Xaa-Thr (in which Xaa is any amino acid except Pro), were inserted into the gp120 sequence so as to incorporate glycan moieties at select positions on the envelope glycoprotein in order to mask non-neutralizing epitopes (Pantophlet et al., 2003b
) (Figure 1). Incorporation of these additional glycans did indeed abolish binding of a broad panel of non- and weakly neutralizing anti-gp120 antibodies (Pantophlet et al., 2003b
). Although b12 binding was preserved, the binding affinity relative to wild-type gp120 was reduced. Furthermore, binding was still observed with antibodies to epitopes in the N-terminus of gp120 (Pantophlet et al., 2003b
).
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A total of 23 mAbs and one polyclonal immunoglobulin preparation (HIVIG) were tested. In addition to antibodies (n = 19) from our previous study (Pantophlet et al., 2003b), mAbs 39F and CO11 (kindly provided by J.Robinson) and mAb F425 B4e8 (kindly provided by L.Cavacini) were also used here. The latter three antibodies are V3-loop specific (Grundner et al., 2002
; Cavacini et al., 2003
). We also included mAb 8.22.2 (kindly provided by A.Pinter), which binds to the V2 loop of gp120 (He et al., 2002
). These four mAbs neutralize primary HIV-1 isolates weakly (Grundner et al., 2002
; He et al., 2002
; Cavacini et al., 2003
). Affinity-purified polyclonal antibody against the C5 region of gp120, which binds the same epitope (APTKAKRRVVQREKR) at the extreme C-terminal region of gp120 as recognized by mAb D7324 (Wyatt et al., 1995
), was purchased from Cliniqa (Fallbrook, CA).
MAb biotinylation
MAb 2G12 was biotinylated using N-hydroxysuccinimidobiotin (Sigma) according to the manufacturer's instructions.
Plasmids and mutagenesis
The generation of plasmid pCMV-Tag4A-tpa was described in an earlier study (Pantophlet et al., 2003a). The plasmid was derived from plasmid pCMV-Tag4A (Stratagene) and contains a tissue plasminogen activator (tpa) leader sequence for extracellular expression of recombinant proteins. The generation of plasmid pCMV-Tag4A-tpaJR-FLgp120-mCHO-GDMR, which encodes the hyperglycosylated gp120 mutant that was previously termed mCHO*-GDMR and is now designated mCHO-GDMR, has been described recently (Pantophlet et al., 2003b
); the parental gp120 sequence of this mutant is that of the HIV-1 primary isolate JR-FL, which was codon-optimized for high-level expression in mammalian cells (Haas et al., 1996
; Andre et al., 1998
). Site-directed mutagenesis to revert previous mutations to wild-type sequence and to introduce additional N-glycosylation signal sequences into mCHO-GDMR was performed using QuikChange (Stratagene).
To generate gp120-containing deletions of residues in the N and C-termini (52 and 19 residues, respectively), we performed PCR using the primers flCORE-5 (5'-GGAGGTCAACAGCACCGCGCGCGAGGTGGTGCTGGAGAATGTGAC-3'), which contains a BssHII restriction site (underlined) and flCORE-3 (5'-GGAGGTCAACAGCACCCTCGAGTTAATTAATTAACTCAATCTTCACCACCTTGTA-3'), which contains a XhoI site (underlined). To truncate only the gp120 N-terminus, the primers flCORE-5 and T7 (5'-TAATACGACTCACTATAGGG-3') were used. The plasmid pCMV-Tag4A-tpaJR-FLgp120-mCHO-GDMR was used as template in all cases. The PCR products were cloned into pCMV-Tag4A-tpa according to standard protocols after BssHII/XhoI restriction enzyme digestion. All plasmids were sequenced prior to usage to ensure that the introduced mutations were correct.
Expression of recombinant HIV glycoproteins
For the expression of wild-type monomeric gp120 and mutant glycoproteins, 293T cells were transiently transfected with the respective envelope plasmids, as described previously (Pantophlet et al., 2003a,b
). Two days post-transfection, culture supernatants containing recombinant glycoproteins were pooled (if necessary) and stored at 20°C until needed.
Enzyme-linked immunosorbent assay (ELISA)
Enzyme immunoassays were performed essentially as described (Pantophlet et al., 2003b). Briefly, glycoproteins were captured on ELISA plate wells using the anti-C5 polyclonal antibody preparation, unless indicated otherwise. Antibodies against CD4-induced epitopes were tested in the absence of soluble CD4. MAb 2G12, or in some cases cyanovirin (CVN) (Boyd et al., 1997
), was used to ensure that similar amounts of envelope proteins were captured in each experiment. In general, plates were developed with p-nitrophenyl phosphate (Sigma) and absorbance was measured at 405 nm. When peroxidase-conjugated secondary antibody (Pierce) was used, plates were developed with 3,3',5,5' tetramethylbenzidine/hydrogen peroxide substrate (TMB/H2O2; Pierce) and absorbances measured at 450 nm. In this case, the color reaction was stopped with sulfuric acid (2 M) prior to spectrophotometric measurement. For detection of biotinylated 2G12, peroxidase-conjugated avidin (Pierce) was used in combination with the TMB system. All assays were performed in duplicate. Apparent binding affinities were calculated as the antibody concentration at half-maximal binding.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a recent study (Pantophlet et al., 2003b), we reported on a gp120 mutant, termed mCHO-GDMR, containing four alanine-substituted residues (G473A, D474A, M475A and R476A) at the center of the CD4-binding site and seven extra N-glycosylation sites, which were incorporated to mask non-neutralizing epitopes with carbohydrates. Mutant mCHO-GDMR and variants thereof are of particular interest for immunogenicity studies because most non-neutralizing mAbs do not bind this antigen, whereas the broadly neutralizing antibody b12 can still bind (Pantophlet et al., 2003b
). However, despite the unique antigenic properties of mutant mCHO-GDMR, the efficacy of this antigen to induce b12-like antibodies may be restricted because b12 binds this mutant with an apparent affinity that is lower than the corresponding affinity for wild-type gp120 and also because binding of a few non-neutralizing gp120 mAbs to mutant mCHO-GDMR was still observed (Pantophlet et al., 2003b
). In the present study, we sought to correct these potential limitations to improve the antigenic quality of this mutant as a b12-tailored antigen.
First, added N-glycans that might potentially be involved in reducing the binding of b12 to mutant mCHO-GDMR were identified by reverting the newly introduced glycosylation sites on the gp120 core to wild-type sequence (see Figure 1); glycosylation sites on the core were reverted first because the inserted glycans are relatively close to the putative b12-binding site on gp120 (Saphire et al., 2001). Reverting the glycosylation signal sequence inserted at positions 9294 to wild-type sequence (designated variant H92Nx; in this study, the suffix x denotes the reversion of an added glycosylation site to wild-type sequence), which lies closest to the putative b12 epitope, resulted in a slight increase in b12-binding affinity relative to mutant mCHO-GDMR but did not improve b12 binding affinity to wild-type levels (Figure 2). To determine whether reverting additional N-glycosylation signal sequences might further rescue b12-binding without loss of the epitope-masking properties of mutant mCHO-GDMR, we tested a small panel of non- or weakly neutralizing CD4bs antibodies and mAb b12 for binding to glycosylation site variants of mutant mCHO-GDMR. We noted that reversion of additional glycosylation sites on the gp120 core did not improve b12 affinity further (Figure 3). Reassuringly, however, reversion of the added N-glycan attachment site at position 92 did not lead to enhanced binding of non-neutralizing anti-CD4bs antibodies tested here (Figure 3A). Significantly increased binding was also not observed for these non-neutralizing CD4bs antibodies with a variant in which the added glycosylation sites at positions 92 and 114 were simultaneously reverted to wild-type sequence (Figure 3B). However, when all three glycosylation sites surrounding the CD4bs were reverted to wild-type sequence (variant H92Nx/Q114Nx/I423Nx), binding of mAbs 15e and F91 was no longer completely abolished (Figure 3C). Given that reversion of the N-glycosylation site at position 92 allowed better b12 binding without affecting the epitope masking properties of the parental mutant, this variant, designated H92Nx, was selected as a template for further mutagenesis.
|
|
To identify regions on gp120 that might be masked by the incorporation of additional glycans on to gp120, we tested mAbs to the V2 loop, to CD4-induced (CD4i) epitopes and to the V3 loop with variants of mutant mCHO-GDMR in which added glycosylation signal sequences were reverted to wild-type sequence. Of the two anti-V2 loop antibodies tested here, mAbs 8.22.2 and G3-4, only the former bound the variant (K171Nx) in which the glycosylation site in the V2 loop was reverted to wild-type sequence (Figure 4A). This result suggested that one or more glycans that had been introduced elsewhere contributed to steric hindrance of mAb G3-4 to the V2 loop. To investigate further, we generated two additional variants, E150Nx/K171Nx and P313Nx/K171Nx, in which the glycosylation sites that were inserted in the V1 loop (E150N) and the V3 loop (P313N), respectively, were reverted to wild-type sequence in the background of the K171Nx variant. We observed that by reverting the added glycosylation site at position 150, the apparent binding affinity of mAb G3-4 was improved, whereas reverting the glycosylation site in the V3 loop did not enhance G3-4 binding (Figure 4B). However, binding of G3-4 to variant E150Nx/K171Nx still did not achieve wild-type levels, and an explanation for this effect is not readily apparent.
|
|
|
Although removal of the glycosylation site at position 92 improved b12-binding affinity relative to the parental hyperglycosylated mutant mCHO-GDMR, antibodies to the C1 region of gp120 were still reactive with this variant (not shown). To abolish binding of these antibodies, the N-terminus of variant H92Nx was truncated up to residue E83, which is analogous to the truncated gp120 construct used for crystallization of the gp120 core (Kwong et al., 1998, 2000
). We reasoned that truncation of the N-terminus would allow for more efficient elimination of several epitopes at once, rather than introducing multiple glycosylation sites in an attempt to eliminate antibody binding to a likely highly flexible N-terminus (Kwong et al., 1999
). ELISA experiments showed that the anti-C1 antibodies tested were indeed not able to bind the variant with the truncated N-terminus, designated
N-mCHO-GDMR (Figure 7A). More importantly, b12 binding was not affected by truncation of the N-terminus. However, as seen previously with mutant mCHO-GDMR (Pantophlet et al., 2003b
), truncation of both N- and C-termini severely diminished b12 binding (Figure 7B).
|
To evaluate the antigenicity of hyperglycosylated variant N-mCHO-GDMR, we tested a panel of 23 mAbs and a polyclonal immunoglobulin preparation by ELISA (Figure 8). Included in this panel were two mAbs, 39F and F425 B4e8, which had not been tested previously with the parental mutant mCHO-GDMR or with any of the variants described so far. Both of these antibodies have been reported to recognize epitopes at the base of the V3 loop (Grundner et al., 2002
; Cavacini et al., 2003
; J.Robinson, personal communication). Only four antibodies were able to bind variant
N-mCHO-GDMR: mAbs 2G12, b12, 39F and F425 B4e8. The fact that the last two V3-loop antibodies were able to bind this variant suggested that the glycan incorporated at position 313 in the apex of the V3 loop was not sufficient to obscure the base of the loop. To eliminate binding of these antibodies, a series of additional hyperglycosylated variants were generated, in which glycosylation signal sequences were added sequentially to the base of the loop. Glycosylation sites were first introduced simultaneously at positions 320 and 325, which are both located at the C-terminus of the V3 loop and then in the V3 loop N-terminal region at residue 306. Binding of mAb F425 B4e8 was diminished by the first two substitutions in the C-terminal end of the base (Figure 9A). Surprisingly, 39F binding was not significantly affected, even when a glycosylation site at position 306 was incorporated (Figure 9B). However, substituting residues at positions 304 (Arg) and 305 (Lys) with Ala completely abolished 39F binding (Figure 9C). These two substitutions also further reduced binding of F425 B4e8 compared with the hyperglycosylated variant containing only the three added glycosylation sites in the V3 loop, suggesting that this antibody may also be interacting with or affected by N-terminal residues at the base of the V3 loop. Taking into consideration that the single glycan at the apex of V3 was insufficient to prevent binding of mAbs directed to the base of the loop and given that the V2 loop in our hyperglycosylated variants contained only one extra glycan (at position 171), a second N-glycosylation site was inserted in the V2 loop at position 180 in variant
N-mCHO-GDMR containing the additional modifications in V3. The resulting variant was designated
N2-mCHO-GDMR. Introduction of the additional glycosylation site in V2 had no effect on b12 binding (data not shown).
|
|
Given the superior antigenic properties of variant N2-mCHO-GDMR relative to the parental hyperglycosylated mutant mCHO-GDMR, we next compared b12 affinity for our hyperglycosylated variants with that of the parental mutant and wild-type gp120, to determine whether we had also succeeded in improving b12 binding relative to the original mutant (Figure 10). As reported previously, the apparent b12 binding affinity was 5-fold lower for mutant mCHO-GDMR than for wild-type gp120 (Pantophlet et al., 2003b
). In comparison, the apparent antibody affinity for variant
N-mCHO-GDMR was equivalent to that for wild-type gp120. For variant
N2-mCHO-GDMR, a slight decrease in b12 affinity was observed relative to wild-type gp120. However, b12 affinity for variant
N2-mCHO-GDMR was higher than the antibody affinity for the parental mutant, indicating that, in addition to enhancing the antigenic qualities of the original mutant, we had also succeeded in improving b12 binding.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To address the first goal, the added glycosylation sites on the gp120 core in mutant mCHO-GDMR were reverted to wild-type sequence. Reverting a single glycosylation site, at position 92, was sufficient to enhance b12 affinity relative to its affinity for the parental hyperglycosylated mutant (Figure 2). Notably, removal of the other two added N-glycosylation sites on the gp120 core at positions 114 and 423, combined with the removal of the added glycosylation site at position 92, did not improve b12 binding further (Figure 3). Hence the decreased b12 affinity for mutant mCHO-GDMR appears to be due either to removal of an antibody contact residue as a result of the introduced glycosylation site, to localized conformational changes surrounding the CD4bs associated with the mutations needed to introduce the glycosylation site or through incorporation of the glycan itself, which may hinder b12 binding. Although it is not obvious at present which of these three possibilities is most likely, results from a previous study suggest that alterations in this region may negatively affect b12 binding (Pantophlet et al., 2003a). For two other anti-CD4bs antibodies, 15e and F91, binding was nearly fully restored only when all three added glycosylation sites on the gp120 core were reverted simultaneously to wild-type sequence, but not when the sites were reverted individually (Figure 3 and data not shown). As it seems unlikely that all three glycosylation sites harbor antibody contact residues, the most likely explanation in this case for the observed increase in 15e and F91 binding is that the incorporated carbohydrate moieties restrict accessibility of these antibodies to their respective epitope. We also observed that at least some of the added glycans have the potential to mask vicinal epitopes. For example, the anti-V2 loop mAb G3-4 was unable to bind its putative epitope [162TTSIRDEVQKEYALFYKLDV181 in gp120JR-FL (Poignard et al., 1996
)] even when the extra glycan incorporated at position 171 in the V2 loop was removed (Figure 4A). However, when the extra glycosylation site at position 150 in the V1 loop was reverted to wild-type sequence, G3-4 binding affinity for gp120 improved (Figure 4B). These observations substantiate the generally accepted notion that the V1 and V2 loops are in close proximity to each other on monomeric gp120 and probably even more so on the functional virion spike (Kwong et al., 1998
; Wyatt et al., 1998
). The second anti-V2 loop antibody, mAb 8.22.2, was able to bind the variant with the reverted V2-glycosylation site, indicating that the masking of neighboring epitopes by the added glycans may in some cases only be partial.
In contrast, the observed inhibition of binding of mAbs CO11 and 447-52D to mutant mCHO-GDMR appears not to be due solely to the presence of the added glycan at position 313, but to substitution of critical antibody contact residues in the apex of the loop, in particular for mAb 447-52D (Gorny et al., 1992; Stanfield et al., 2004
). Whether this is also true for mAb CO11 is currently not assessable because the epitope recognized by this antibody has not been mapped. In addition to substituting crucial antibody contact residues, mutation of residues P313 and/or R315 may also cause an alteration in the type I and II ß-turns that are characteristic of the apex of the V3 loop and are required for proper presentation of the antibody epitopes (Stanfield et al., 2003
). We make particular note here of the specific elimination of the 447-52D epitope. This antibody neutralizes
50% of clade B isolates (Binley et al., 2004
) and is therefore considered a somewhat broader neutralizing antibody than other typical V3 loop antibodies with only limited neutralizing capacities (Kwong, 2004
). However, we can accept the loss of 447-52D binding in the context of our hyperglycosylation strategy, given that our goal is to obtain b12-like antibodies which, ideally, would have broader cross-clade neutralizing properties than those exhibited by mAb 447-52D.
Having improved binding of b12 relative to mutant mCHO-GDMR, we next sought to improve the antigenicity of the hyperglycosylated variant H92Nx. First, we truncated the N-terminus of gp120 up to residue E83 to abolish binding of non-neutralizing anti-C1 antibodies (Figure 7). Binding of b12 to the resulting variant, designated N-mCHO-GDMR, was not negatively affected. However, b12 binding was severely reduced when both N- and C-termini were truncated. Although we have not explored whether truncation of the C-terminus alone affects b12 binding, the above results indicate that the C-terminal region of gp120 may play a role in the conformational integrity of the b12 epitope. ELISA experiments with a panel of more than 20 antibodies showed that variant
N-mCHO-GDMR had not lost the epitope-masking properties of the parental mutant (Figure 8).
However, two anti-V3 loop antibodies, F425 B4e8 and 39F, which had not been tested previously, were able to bind variant N-mCHO-GDMR. Hence the antigenic properties of this variant needed to be improved further, given that the single glycan incorporated in the apex of the V3 loop was insufficient to mask the entire loop. Similar observations have been made in a previous study (Garrity et al., 1997
) in which immunogenicity studies showed that incorporation of a glycan on the C-terminal side of V3 caused a shift in antibody response to the N-terminal side of the loop, whereas the opposite effect was observed when a glycan was incorporated at the N-terminal side. Such findings illustrate the remarkable inherent antigenicity and immunogenicity of the V3 loop. To address this problem here, we inserted further N-glycosylation sites at the base of the V3 loop to mask the putative epitopes of antibodies F425 B4e8 and 39F. Although this strategy proved successful for F425 B4e8, two alanine substitutions, R304A and K305A, were required in addition to the incorporated glycans to eliminate binding of mAb 39F (Figure 9).
We also inserted an additional glycosylation site in the V2 loop, at position 180, to mask epitopes that might not be covered by the single glycan at position 171 that was incorporated into the parental mutant. We termed our newly generated hyperglycosylated variant N2-mCHO-GDMR. This variant only binds the broadly neutralizing mAbs b12 and 2G12 and a monospecific antibody preparation against the extreme C-terminal region of gp120 (Table I). Although one could argue that the C-terminus may also elicit unwanted non-neutralizing antibodies, essentially all antibodies to the C5 region that have been characterized to date appear to preferentially bind denatured gp120 (http://www.hiv.lanl.gov/content/immunology/index.html), indicating that anti-C5 antibodies may not be elicited readily when immunizations are performed with native gp120 formulated in a non-denaturing adjuvant preparation.
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barouch,D.H. and Letvin,N.L. (2002) Vaccine, 20, Suppl. 4, A66A68.[CrossRef][ISI][Medline]
Binley,J.M. et al. (2004) J. Virol., 78, 1323213252.
Boyd,M.R. et al. (1997) Antimicrob. Agents Chemother., 41, 15211530.[Abstract]
Burton,D.R. (2002) Nat. Rev. Immunol., 2, 706713.[CrossRef][ISI][Medline]
Burton,D.R., Barbas,C.F., 3rd, Persson,M.A., Koenig,S., Chanock,R.M. and Lerner,R.A. (1991) Proc. Natl Acad. Sci. USA, 88, 1013410137.[Abstract]
Burton,D.R. et al. (1994) Science, 266, 10241027.[ISI][Medline]
Burton,D.R., Desrosiers,R.C., Doms,R.W., Koff,W.C., Kwong,P.D., Moore,J.P., Nabel,G.J., Sodroski,J.G., Wilson,I.A. and Wyatt,R.T. (2004) Nat. Immunol., 5, 233236.[CrossRef][ISI][Medline]
Calarese,D.A. et al. (2003) Science, 300, 20652071.
Cavacini,L., Duval,M., Song,L., Sangster,R., Xiang,S.H., Sodroski,J. and Posner,M. (2003) AIDS, 17, 685689.[CrossRef][ISI][Medline]
D'Souza,M.P., Livnat,D., Bradac,J.A. and Bridges,S.H. (1997) J. Infect. Dis., 175, 10561062.[ISI][Medline]
Garrity,R.R., Rimmelzwaan,G., Minassian,A., Tsai,W.P., Lin,G., de Jong,J.J., Goudsmit,J. and Nara,P.L. (1997) J. Immunol., 159, 279289.[Abstract]
Gavel,Y. and von Heijne,G. (1990) Protein Eng., 3, 433442.[ISI][Medline]
Gorny,M.K., Conley,A.J., Karwowska,S., Buchbinder,A., Xu,J.-Y., Emini,E.A., Koenig,S. and Zolla-Pazner,S. (1992) J. Virol., 66, 75387542.[Abstract]
Graham,B.S. (2002) Annu. Rev. Med., 53, 207221.[CrossRef][ISI][Medline]
Grundner,C., Mirzabekov,T., Sodroski,J. and Wyatt,R. (2002) J. Virol., 76, 35113521.
Haas,J., Park,E.C. and Seed,B. (1996) Curr. Biol., 6, 315324.[ISI][Medline]
He,Y., Honnen,W.J., Krachmarov,C.P., Burkhart,M., Kayman,S.C., Corvalan,J. and Pinter,A. (2002) J. Immunol., 169, 595605.
Kasturi,L., Chen,H. and Shakin-Eshleman,S.H. (1997) Biochem. J., 323, 415419.[ISI][Medline]
Kwong,P.D. (2004) Structure, 12, 173174.[ISI][Medline]
Kwong,P.D., Wyatt,R., Robinson,J., Sweet,R.W., Sodroski,J. and Hendrickson,W.A. (1998) Nature, 393, 648659.[CrossRef][ISI][Medline]
Kwong,P.D, Wyatt,R., Desjardins,E., Robinson,J., Culp,J.S., Hellmig,B.D., Sweet,R.W., Sodroski,J. and Hendrickson,W.A. (1999) J. Biol. Chem., 274, 41154123.
Kwong,P.D., Wyatt,R., Majeed,S., Robinson,J., Sweet,R.W., Sodroski,J. and Hendrickson,W.A. (2000) Struct. Fold. Des., 8, 13291339.[Medline]
Labrijn,A.F. et al. (2003) J. Virol., 77, 1055710565.
Lee,H.K., Scanlan,C.N., Huang,C.Y., Chang,A.Y., Calarese,D.A., Dwek,R.A., Rudd,P.M., Burton,D.R., Wilson,I.A. and Wong,C.H. (2004) Angew. Chem. Int. Ed., 43, 10001003.[CrossRef][ISI]
Letvin,N.L., Barouch,D.H. and Montefiori,D.C. (2002) Annu. Rev. Immunol., 20, 7399.[CrossRef][ISI][Medline]
Li,H. and Wang,L.X. (2004) Org. Biomol. Chem., 21, 483488.[CrossRef]
McMichael,A.J. and Hanke,T. (2003) Nat. Med., 9, 874880.[CrossRef][ISI][Medline]
Moore,J.P. and Sodroski,J. (1996) J. Virol., 70, 18631872.[Abstract]
Moulard,M. et al. (2002) Proc. Natl Acad. Sci. USA, 99, 69136918.
Pantophlet,R. and Burton,D.R. (2003) Trends Mol. Med., 9, 468473.[CrossRef][ISI][Medline]
Pantophlet,R., Ollmann Saphire,E., Poignard,P., Parren,P.W., Wilson,I.A. and Burton,D.R. (2003a) J. Virol., 77, 642658.[CrossRef][ISI][Medline]
Pantophlet,R., Wilson,I.A. and Burton,D.R. (2003b) J. Virol., 77, 58895901.
Parren,P.W., Marx,P.A., Hessell,A.J., Luckay,A., Harouse,J., Cheng-Mayer,C., Moore,J.P. and Burton,D.R. (2001) J. Virol., 75, 83408347.
Poignard,P., Fouts,T., Naniche,D., Moore,J.P. and Sattentau,Q.J. (1996) J. Exp. Med., 183, 473484.[Abstract]
Poignard,P., Saphire,E.O., Parren,P.W. and Burton,D.R. (2001) Annu. Rev. Immunol., 19, 253274.[CrossRef][ISI][Medline]
Rizzuto,C. and Sodroski,J. (2000) AIDS Res. Hum. Retroviruses, 16, 741749.[CrossRef][ISI][Medline]
Rizzuto,C.D., Wyatt,R., Hernandez-Ramos,N., Sun,Y., Kwong,P.D., Hendrickson,W.A. and Sodroski,J. (1998) Science, 280, 19491953.
Roben,P., Moore,J.P., Thali,M., Sodroski,J., Barbas,C.F.,III and Burton,D.R. (1994) J. Virol., 68, 48214828.[Abstract]
Sanders,R.W., Venturi,M., Schiffner,L., Kalyanaraman,R., Katinger,H., Lloyd,K.O., Kwong,P.D. and Moore,J.P. (2002) J. Virol., 76, 72937305.
Saphire,E.O., Parren,P.W., Pantophlet,R., Zwick,M.B., Morris,G.M., Rudd,P.M., Dwek,R.A., Stanfield,R.L., Burton,D.R. and Wilson,I.A. (2001) Science, 293, 11551159.
Sattentau,Q.J., Zolla-Pazner,S. and Poignard,P. (1995) Virology, 206, 713717.[ISI][Medline]
Sattentau,Q.J., Moulard,M., Brivet,B., Botto,F., Guillemot,J.C., Mondor,I., Poignard,P. and Ugolini,S. (1999) Immunol. Lett., 66, 143149.[CrossRef][ISI][Medline]
Sayle,R.A. and Milner-White,E.J. (1995) Trends Biochem. Sci., 20, 374.[CrossRef][ISI][Medline]
Scanlan,C.N., Pantophlet,R., Wormald,M.R., Ollmann Saphire,E., Stanfield,R., Wilson,I.A., Katinger,H., Dwek,R.A., Rudd,P.M. and Burton,D.R. (2002) J. Virol., 76, 73067321.
Stanfield,R.L., Ghiara,J.B., Ollmann Saphire,E., Profy,A.T. and Wilson,I.A. (2003) Virology, 315, 159173.[CrossRef][ISI][Medline]
Stanfield,R.L., Gorny,M.K., Williams,C., Zolla-Pazner,S. and Wilson,I.A. (2004) Structure, 12, 193204.[ISI][Medline]
Thali,M., Moore,J.P., Furman,C., Charles,M., Ho,D.D., Robinson,J. and Sodroski,J. (1993) J. Virol., 67, 39783988.[Abstract]
Trkola,A., Purtscher,M., Muster,T., Ballaun,C., Buchacher,A., Sullivan,N., Srinivasan,K., Sodroski,J., Moore,J.P. and Katinger,H. (1996) J. Virol., 70, 11001108.[Abstract]
Veazey,R.S. et al. (2003) Nat. Med., 9, 343346.[CrossRef][ISI][Medline]
Wu,L. et al. (1996) Nature, 384, 179183.[CrossRef][ISI][Medline]
Wyatt,R., Moore,J., Accola,M., Desjardin,E., Robinson,J. and Sodroski,J. (1995) J. Virol., 69, 57235733.[Abstract]
Wyatt,R., Kwong,P.D., Desjardins,E., Sweet,R.W., Robinson,J., Hendrickson,W.A. and Sodroski,J.G. (1998) Nature, 393, 705711.[CrossRef][ISI][Medline]
Wyatt,R. and Sodroski,J. (1998) Science, 280, 18841888.
Xiang,S.H., Doka,N., Choudhary,R.K., Sodroski,J. and Robinson,J.E. (2002) AIDS Res. Hum. Retroviruses, 18, 12071217.[CrossRef][ISI][Medline]
Zhu,X., Borchers,C., Bienstock,R.J. and Tomer,K.B. (2000) Biochemistry, 39, 1119411204.[CrossRef][ISI][Medline]
Received October 12, 2004; accepted October 15, 2004.
Edited by James Marks