Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Nigel J. Dimmock
n.j.dimmock{at}warwick.ac.uk
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ABSTRACT |
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These authors contributed equally to this work.
Present address: Health Protection Agency, Porton Down, Salisbury SP4 0JG, UK.
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INTRODUCTION |
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MicroAbs have proved difficult to identify. This may be because the potential MicroAb does not mimic the conformation of the CDR sufficiently well, because effective IgG binding requires more than one CDR or because the mass of the IgG is essential to its biological activity. Nonetheless, several MicroAbs that bind to their cognate antigens have been synthesized (Williams et al., 1988, 1989a
, b
, 1991a
, b
; Cohen et al., 1990
; Welling et al., 1990
, 1991
; Winter & Milstein, 1991
; Jarrin et al., 1994
; Smith et al., 1994
; Laune et al., 1997
; Monnet et al., 1999
), but so far only three virus-neutralizing MicroAbs have been isolated. The first was derived from the HIV-1 gp120 envelope glycoprotein-specific mAb F58 (Broliden et al., 1990
; Levi et al., 1993
). This 17-mer (CDLIYYDYEEDYYFDYC) binds to soluble gp120 and virions and neutralizes infectivity. Its neutralization increases when it is disulfide cyclized through the terminal cysteine residues (Levi et al., 1993
). Both the IgG and MicroAb recognize the same epitope in the tip of the V3 loop. This is RKxxxI**GPGR, where x is any amino acid residue and ** are residues absent from some HIV-1 strains that do not appear to influence neutralization by F58 (Broliden et al., 1991
; Miller et al., 1998
). On a molar basis, the MicroAb neutralizes HIV-1 strain IIIB 32-fold less efficiently than the IgG (Jackson et al., 1999
). The second neutralizing MicroAb was derived from CDR-H3 of mAb RS-348 to the F glycoprotein of human respiratory syncytial virus (Bourgeois et al., 1998
). This comprises 21 CDR residues with an extra cysteine at each terminus and has about 1000-fold less activity than the IgG. The linear form has more neutralizing activity than the cyclic form at low concentration and equal activity at high concentration. It also protected mice from respiratory disease in vivo when administered intranasally. A third MicroAb of 27 residues is based on the contact residues of five of the CDRs of mAb ST40 that recognizes the HIV-1 primary receptor CD4; it blocked HIV-1 replication rather inefficiently (Casset et al., 2003
).
Attempts have been made to increase the neutralizing activity of F58 MicroAb by fusing it with a protein carrier in order to retain a more cogent conformation, increase valency with multiple copies and increase molecular mass. This has not been successful (Fontenot et al., 1996, 1998
). However, a MicroAb derived from the highly active gp120-specific mAb b12 neutralized HIV-1 when fused with BSA, but was still 106-fold less active than the IgG (Saphire et al., 2001
; D. R. Burton, personal communication).
This report investigates the interactions of the F58 MicroAb with its epitope in the V3 loop of the gp120 envelope glycoprotein of HIV-1. As the one time principal neutralizing determinant, the V3 loop has been much studied, with early work suggesting that the disulfide bonded loop was relatively unstructured, that cognate antibodies were strain specific and effective only against T-cell-line-adapted viruses, and that the V3 loop acted as an immunological decoy that underwent extensive mutation under positive immune selection (Levy, 1998; Poignard et al., 2001
). Some V3 epitopes may be affected allosterically by binding of CD4-binding site antibodies, supporting the structured nature of the loop (e.g. Thali et al., 1992
; Pinter et al., 1993
). V3 antibodies that neutralize primary virus isolates have now been found (Krachnarov et al., 2001
; Gorny et al., 2002
, 2004
). The functions of the V3 loop are manifold. While most V3-specific antibodies do not inhibit attachment to the CD4 primary receptor, some do (Edinger et al., 2000
), indicating that the V3 loop and/or attached IgG interfere sterically with the CD4 receptor-binding site. Other data suggest that the V3 loops of both cell-line-adapted and primary virus isolates interact sterically with the CD4-binding site (e.g. Moore et al., 1993
; Javaherian et al., 1994
; Mbah et al., 2001
; Zhang et al., 2002
) and are involved in binding the co-receptors, CCR5 and CXCR4 (Rizzuto et al., 1998
; Basmaciogullari et al., 2002
). This could explain the observed fusion-inhibiting activity of the F58 MicroAb (Jackson et al., 1999
). The V3 loop also interacts with the C2 and V1/V2 regions of gp120 (Willey et al., 1989
; Cao et al., 1997
; Labrosse et al., 2001
; Losman et al., 2001
; Hoffman et al., 2002
), ganglioside GM3, glycosphingolipids (Hammache et al., 1998
; Nehete et al., 2002
) and some host cell proteins (Minder et al., 2002
). The V3 loop functions in cell tropism through its co-receptor binding activity (Hoffman & Doms, 1999
).
Here, alanine scanning analysis, mass spectroscopy (MS) and surface plasmon resonance were used to investigate the interaction of F58 MicroAb with its epitope expressed on gp120 peptides, the gp120 protein monomer and the gp120gp41 oligomer on the surface of HIV-1 virions.
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METHODS |
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Virus assay and neutralization.
Virus infectivity was assayed by the production of syncytia in C8166 cell monolayers. There is a linear relationship between the number of syncytia and the amount of inoculum, meaning that each syncytium is the product of a single infectious unit (McLain & Dimmock, 1994). The assay thus reflects a single cycle of replication. Plates (96-well; Gibco-BRL) were treated with poly-L-lysine (50 µg ml1; Sigma) to anchor cells and seeded with 5x104 C8166 cells per well. Cells were inoculated with approximately 50 syncytium-forming units (s.f.u.) per well for 1 h at 37 °C. After removing virus and rinsing cells, medium (200 µl per well; 6 replicates) was added. The plate was incubated for 3 days and syncytia were counted. Although this was a 3-day assay, secondary syncytia do not appear until later (McLain & Dimmock, 1994
). Syncytia contain three or more nuclei and their identity was confirmed by cytostaining. However, they are readily recognized under low power microscopy. The same virus titre is obtained when culture fluid p24 is assayed by ELISA (data not shown). For neutralization, medium, MicroAb or antibody in half log10 dilutions, was mixed with an equal volume of 2000 s.f.u. HIV-1 ml1 for 1 h at 37 °C and inoculated as above. The virusantibody mix was then replaced with medium. The virus control gives approximately 50 syncytia per well. Neutralization is expressed as a percentage loss of syncytia compared with the virus control and curves were calculated with Prism software. An interpolated 75 % neutralization point was chosen for calculating neutralization relative to that achieved by wild-type (wt) MicroAb, taken as 100 %).
Antibodies and peptide antigens.
The 17 residue MicroAb (CDLIYYDYEEDYYFDYC) is derived from CDR-H3 of HIV-1 gp120 V3 loop-specific F58 murine IgG1 (Levi et al., 1993). This MicroAb and the alanine-substituted MicroAbs were synthesized by Sigma Genosys UK. All peptides were cyclized by a terminal disulfide bond. Purity, as judged by MS, was >95 % for most MicroAbs; purity of the others was >89 % (MicroAbs 3, 8, 16), 80 % (10), 76 % (11) and 45 % (15). Due to production difficulties, there was only a low yield of MicroAb 16. The V3 peptides used were a 17 residue linear peptide (Z321; RKSISIGPGRAFFATGD) from an African clade A primary isolate and a 35-residue cyclized BRA peptide (CTRPNNNTRKSIHIGPGRAFYATGEIIGDIRQAHC) from a Brazilian clade B primary isolate. BRA comprises the entire V3 loop and has a terminal disulfide bond. These peptides were supplied by the Centralized Facilities for AIDS Reagents (ADP 765 and EVA 7026, respectively). Both express the F58 IgG and MicroAb epitope sequence, RKxxxIGPGR (Millar et al., 1998
). Soluble recombinant gp120 (>90 % glycosylated) from HIV-1 IIIB was produced in Chinese hamster ovary cells (Centralized Facilities for AIDS Reagents). F58 IgG was purified using Protein G-Sepharose CL-4B (Sigma).
Binding of alanine-substituted MicroAb to gp120.
This assay determined the ability of alanine-substituted F58 MicroAbs to compete with the binding of F58 IgG to soluble gp120. A 96-well plate (Immulon 2; Dylan) was incubated with soluble gp120 (0·01 µg per well) in coating buffer (0·1 M NaHCO3, pH 8·5) or buffer alone. All incubations were at room temperature unless stated. Plates were washed with TBS-T (150 mM Tris, 140 mM NaCl, pH 7·6, 0·05 % Tween 20) and blocked with 3 % BSA in TBS-T for 2 h. After further washing, 100 µl MicroAb per well (0, 0·3, 1, 3, 10 µg per well) was added and plates were incubated overnight. After washing, mAb F58 (0·01 µg per well) or TBS-T was added for 2 h. Plates were washed and then incubated successively with optimized concentrations of biotinylated anti-mouse IgG (Amersham Life Science) and streptavidin-conjugated alkaline phosphatase (Amersham Life Science) both for 1 h. Finally, they were incubated with p-nitrophenyl phosphate in diethanolamine buffer (Pierce & Warriner) at 37 °C and the OD405 was measured. Inhibition of mAb F58 binding by MicroAb is expressed as a percentage of F58 binding in the absence of MicroAb and curves were calculated by Prism software. The 25 % inhibition point was chosen for calculating binding relative to the binding of wt MicroAb (100 %) as this gave the greatest discrimination between peptides. Affinities were calculated from the amount of each MicroAb required to give 50 % inhibition of binding of F58 IgG.
Reduction and alkylation of the MicroAb disulfide bond.
MicroAb (50 µg) was dissolved in 200 µl buffer (0·5 M Tris/HCl, 6 M guanidineHCl, 0·02 M EDTA, pH 8·5). Dithiothreitol was added to the Tris buffer to a concentration of 4·8 mM and the mixture was incubated at 50 °C for 4 h. After cooling, 20 µl 100 mM iodoacetic acid in Tris buffer was added for 1 h at 37 °C. Finally, reduced and alkylated MicroAb (Mr 2407·8) was purified on a reversed phase micro-column (Zip-tip; Millipore).
Hydrogen/deuterium (H/D) exchange.
Individual solutions of MicroAb, reduced MicroAb, Z321 and BRA (all approximately 20 µM) were lyophilized and redissolved in D2O (99·96 % D; Sigma) at room temperature. Aliquots were rapidly transferred to the mass spectrometer and H/D exchange rates were measured. Complexes were formed by incubating 200 µM MicroAb and 20 µM Z321 or BRA in H2O at room temperature for 30 min. H/D exchange was initiated by diluting 1 : 10 with D2O and mass spectra were recorded with time. Complexes were also formed with deuterated MicroAb and deuterated Z321 or BRA.
Mass Spectrometry (MS).
Nano-electrospray mass spectra were recorded on a Q-TOF quadrupole/orthogonal time-of-flight MS (Micromass UK). For recording mass spectra, the quadrupole was operated in radio frequency mode and all transmitted ions were accelerated orthogonally into the time-of-flight mass analyser.
Surface plasmon resonance analysis of MicroAbgp120 complexes.
Real-time interactions between solid phase MicroAb and soluble gp120 were studied using the BIAcore 2000 system. Biotinylated MicroAb (the biotin is N-terminal) was diluted in HBS-EP buffer pH 7·4 (Biacore) to 0·05 µg µl1 and 300 µl was injected at a flow rate of 10 µl min1 over a streptavidin-coated SA sensor chip. After washing with HBS-EP buffer for 4 h, approximately 278 units MicroAb were captured on the chip. gp120 in HBS-EP buffer (16·7416·5 nM) was injected for 4 min at 30 µl min1. Dissociation of bound gp120 was recorded for 10 min. Between each injection, the antigen was dissociated with 10 mM NaOH. mAb F58 IgG was diluted with acetate buffer (pH 4) and approximately 900 units were immobilized on a CM5 sensor chip by amine coupling. gp120 (16·7167 nM) was injected for 4 min at 30 µl min1, followed by 10 min dissociation in HBS-N buffer.
Infrared (IR) spectroscopy.
Attenuated total reflection (ATR) Fourier transform IR (FTIR) spectra were recorded at room temperature on a Bruker Vector 22 IR spectrometer equipped with a liquid nitrogen-cooled mercury/cadmium/telluride detector at a nominal resolution of 2 cm1 in the range 10004000 cm1. The spectrometer was continuously purged with dried air (Jun-Air 600) to minimize the spectral contribution of atmospheric water. Residual water vapour peaks were subtracted using reference spectra and baseline correction was applied when necessary. An aliquot of 200 µl BRA or Z231 peptide (5 µM) or 10 µl F58 MicroAb (1 mM) in 45 mM MES buffer, pH 7, was deposited on an internal reflection element (Kazlauskaite et al., 2003) and a thin film of hydrated peptide was obtained by slowly evaporating the excess water under a stream of N2 gas. Final spectra are a mean of 256 scans, corrected for the background using a clean germanium plate. ATR FTIR spectra of deuterated samples were collected to aid analysis of protein secondary structure (Goormaghtigh et al., 1990
). Deposited films, prepared from samples in H2O, were subjected to a stream of D2O-saturated N2 gas for 10 min at room temperature. Peak fitting of the amide I band (16001700 cm1) was performed on non-deconvoluted spectra using GRAMS 32/AI software (Thermogalactic). Best fits to the experimental spectra were obtained with a Lorentzian lineshape with a full width at half height 6 cm1. Band assignments were made according to Cabiaux et al. (1989)
.
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RESULTS |
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Analysis of deuterated MicroAbantigen complexes by MS
MicroAbV3 peptide complexes have been analysed previously by ESI-MS and the minimum epitope was determined by protease digestion of the antigen (Millar et al., 1998). Using similar technology, it was confirmed that the MicroAb and Z321 V3 peptide formed a 1 : 1 complex (Fig. 4a
). The mass increase when the aqueous complex was incubated in D2O (Fig. 4b
) equated to 50 deuteriums, the same value as that obtained when deuterated MicroAb and Z321 V3 peptides were reacted together (Fig. 4c
). This suggested that the complex is in equilibrium with free MicroAb and antigen and that the rates of dissociation and complex formation are rapid. However, the rate of H/D exchange of the MicroAbZ321 complex was reproducibly lower than that of the MicroAb alone, with full exchange in the complex taking 7 min (Fig. 5
), showing that some groups were protected by peptide interaction. Essentially the same results were obtained with complexes formed between MicroAb and the larger, cyclized BRA V3 peptide (data not shown).
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DISCUSSION |
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The HIV-1-specific F58 MicroAb is a CDR-H3-derived, terminal cysteine-cyclized, 17 residue peptide. Data obtained here from alanine scanning demonstrate that 11D, 12Y and 13Y are essential for both binding and neutralization (Table 2). Thus, binding to the gp120 monomer appears to reflect the neutralization that is mediated via the virion gp120 trimer. However, alanine-substituted 3L, 8Y and 14F MicroAbs bound efficiently to gp120, but gave little neutralization. This suggests that there is a separation of function within the CDR into residues that are concerned with binding to the epitope and those involved in neutralization. Residues 5Y, 6Y and 9E also functioned mainly in neutralization. It is known from previous work that MicroAb does not prevent attachment of virus to target cells (Jackson et al., 1999
) and that its antiviral activity correlates largely with inhibition of virioncell fusion, possibly because it compromises binding of the V3 loop to the co-receptor (Rizzuto et al., 1998
; Basmaciogullari et al., 2002
). The data are also consistent with the loss of MicroAb neutralizing activity found when Y5 or Y6 was deleted, or in MicroAbs in which a conservative substitution (Y5F or Y6F) was made (Jackson et al., 1999
). Having different regions for binding and neutralization may explain why MicroAb F58 was over 100-fold more effective at neutralizing HIV-1 than the F58 CDR-H3 sequence, 4IYYDYEED11, incorporated into a 60 aa triplet repeat of the MUC-1 protein (Fontenot et al., 1996
), even though the triplet repeat is larger and trivalent. Others have commented on the disproportionately high number of tyrosine residues in CDRs that engage antigen (Davies et al., 1990
). Six of the 17 residues of the F58 MicroAb are tyrosine.
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Surface plasmon resonance determinations with soluble gp120 as antigen demonstrated that immobilized MicroAb (Mr 2293·5) was able to capture gp120, a molecule over 50-fold larger, in a flow system. The expectation that the monovalent antibody and antigen form 1 : 1 complexes was confirmed experimentally. Data also show that the F58 MicroAb, in accord with its small size (1 % of the mass of the IgG), associated with gp120 faster than F58 IgG. The MicroAb also dissociated faster, as expected for a molecule that only has one of the six CDRs of the monovalently binding IgG. Despite this dissociation rate, the MicroAb has an equilibrium (KD) value of 7·5 nM, which is similar or better than the best virus-neutralizing antibodies [e.g. the affinities of FAb b12 with IIIB gp120 and MN gp120 are 6·3 and 44 nM, respectively (Barbas et al., 1994)]. The high affinity of the MicroAb could explain the non-reversibility of HIV-1 neutralization observed when virusMicroAb complexes were separated from free MicroAb and incubated for 5 h before inoculation onto cells (Jackson et al., 1999
). The high KD determined here with gp120 suggests that the failure of the H/D exchange experiments with MicroAbV3 peptide antigen complexes resulted from the V3 peptides presenting the F58 epitope in a suboptimal conformation.
The ability of the MicroAb to neutralize virion infectivity is informative about neutralization in general and neutralization of HIV-1 by F58 IgG in particular. Data show that neutralization via the F58 epitope can be mediated by a monomeric ligand and that cross-linking of structures within the virion is not required. MicroAb F58 does not prevent attachment of virus to the CD4 receptor on the target cell (Jackson et al., 1999). This is expected as the MicroAb attached to the V3 loop does not overlap the CD4-binding site. However, F58 MicroAb-directed neutralization correlated with inhibition of virioncell fusion and fusion of HIV-1-infected to non-infected cells (Jackson et al., 1999
). Burton and colleagues have proposed an occupancy theory of neutralization in which loss of infectivity occurs when sufficient molecules of antibody bind to the surface of a virion to sterically interfere with virion attachment to target cell receptors, or with a post-attachment entry event. For HIV-1, Parren & Burton (2001)
proposed that neutralization requires approximately one IgG molecule per envelope trimer. However, F58 MicroAb is only about 1 % of the mass of an IgG molecule and hence has little capacity for steric inhibition. Thus, the MicroAb appears to bind to, or very close to, a functional region of the loop and neutralize by inhibiting a specific virion function, rather than by exercising the non-specific, steric effects of antibody occupancy.
There is clearly a problem with the structure of the antigen peptides BRA and Z231, despite BRA being disulfide linked, as it is in gp120. They are structurally different, with Z231 being over 30 % -sheet and BRA being 2030 %
-helix and random coil. Both have a high
-turn content (Fig. 6b, c
: band at 1674 cm1). Furthermore, the low intensity of amide II after H/D exchange suggests that these two peptides are less compactly folded than F58 MicroAb. Poor binding of BRA and Z321 with MicroAb as shown by H/D exchange and MS and by tyrosine fluorescence spectroscopy (data not shown) further suggests that neither has achieved the (unknown) structure of the gp120 V3 loop that stably binds MicroAb.
Finally, it has been recently shown that certain virus evasion strategies sterically protect critical areas of the envelope protein of HIV-1, such as the co-receptor binding site, from being accessed by IgG molecules (Labrijn et al., 2003). Microantibodies like F58 may therefore be useful stepping stones towards small molecule therapeutics.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Basmaciogullari, S., Babcock, G. J., van Ryk, D., Wojtowicz, W. & Sodroski, J. (2002). Identification of conserved and variable structures in the human immunodeficiency virus gp120 glycoprotein of importance for CXCR4 binding. J Virol 76, 1079110800.
Bourgeois, C., Bour, J. B., Aho, L. S. & Pothier, P. (1998). Prophylactic administration of a complementarity-determining region derived from a neutralizing monoclonal antibody is effective against respiratory syncytial virus infection in BALB/c mice. J Virol 72, 807810.
Broliden, P.-A., Ljunggren, K., Hinkula, J., Norrby, E., Åkerblom, L. & Wahren, B. (1990). A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization. J Virol 64, 936940.[Medline]
Broliden, P.-A., Makitalo, B., Akerblom, L., Rosen, J., Broliden, K., Utter, G., Jondal, M., Norrby, E. & Wahren, B. (1991). Identification of amino acids in the V3 region of gp120 critical for virus neutralization by human HIV-1-specific antibodies. Immunology 73, 371376.[Medline]
Cabiaux, V., Brasseur, R., Wattiez, R., Falmagne, P., Ruysschaert, J.-M. & Goormaghtigh, E. (1989). Secondary structure of diphtheria toxin and its fragments interacting with acidic liposomes studied by polarized infrared spectroscopy. J Biol Chem 264, 49284938.
Calarese, D. A., Scanlan, C. N., Zwick, M. B. & 13 other authors (2003). Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300, 20652071.
Cao, J., Sullivan, N., Desjardin, E., Parolin, C., Robinson, J., Wyatt, R. & Sodroski, J. (1997). Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein. J Virol 71, 98089812.[Abstract]
Casset, F., Roux, F., Mouchet, P. & 10 other authors (2003). A peptide mimetic of an anti-CD4 monoclonal antibody by rational design. Biochem Biophys Res Commun 307, 198205.[CrossRef][Medline]
Cohen, J. A., Williams, W. V., Weiner, D. B., Geller, H. M. & Greene, M. I. (1990). Ligand binding to the cell surface receptor for reovirus type 3 stimulates galactocerebroside expression by developing oligodendrocytes. Proc Natl Acad Sci U S A 87, 49224926.
Davies, D. R., Padlan, E. A. & Sheriff, S. (1990). Antibodyantigen complexes. Annu Rev Biochem 59, 439473.[CrossRef][Medline]
Edinger, A. L., Ahuja, M., Sung, T., Baxter, K. C., Haggarty, B., Doms, R. W. & Hoxie, J. A. (2000). Characterization and epitope mapping of neutralizing monoclonal antibodies produced by immunization with oligomeric simian immunodeficiency virus envelope protein. J Virol 74, 79227935.
Fontenot, J. D., Zacharopoulos, V. R. & Phillips, D. M. (1996). Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles. J Virol 70, 65576562.[Abstract]
Fontenot, J. D., Tan, X. & Phillips, D. M. (1998). Structure-based design of peptides that recognise the CD4 binding domain of HIV-1 gp120. AIDS 12, 14131418.[CrossRef][Medline]
Goormaghtigh, E., Cabiaux, V. & Ruysschaert, J.-M. (1990). Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier-transform infrared spectroscopy on hydrated films. Eur J Biochem 193, 409420.[Abstract]
Gorny, M. K., Williams, C., Volsky, B. & 8 other authors (2002). Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades. J Virol 76, 90359045.
Gorny, M. K., Revesz, K., Williams, C. & 10 other authors (2004). The V3 loop is accessible on the surface of most human immunodeficiency virus type 1 primary isolates and serves as a neutralization epitope. J Virol 78, 23942404.
Hammache, D., Yahi, N., Piéroni, G., Ariasi, F., Tamalet, C. & Fantini, J. (1998). Sequential interaction of CD4 and HIV-1 gp120 with a reconstituted membrane patch of ganglioside GM3: implications for the role of glycolipids as potential HIV-1 fusion cofactors. Biochem Biophys Res Commun 246, 117122.[CrossRef][Medline]
Hoffman, T. L. & Doms, R. W. (1999). HIV-1 envelope determinants for cell tropism and chemokine receptor use. Mol Membr Biol 16, 5765.[CrossRef][Medline]
Hoffman, N. G., Seillier-Moiseiwitsch, F., Ahn, J. H., Walker, J. M. & Swanstrom, R. (2002). Variability in the human immunodeficiency virus type 1 gp120 Env protein linked to phenotype-associated changes in the V3 loop. J Virol 76, 38523864.
Jackson, N. A. C., Levi, M., Wahren, B. & Dimmock, N. J. (1999). Properties and mechanism of action of a 17 amino acid, V3 loop-specific microantibody that binds to and neutralizes human immunodeficiency virus type 1 virions. J Gen Virol 80, 225236.[Abstract]
Jarrin, A., Andrieux, A., Chapel, A., Buchou, T. & Marguerie, G. (1994). A synthetic peptide with anti-platelet activity derived from a CDR of an anti-GPIIb-IIIa antibody. FEBS Lett 354, 169172.[CrossRef][Medline]
Javaherian, K., Langlois, A. J., Montefiori, D. C. & 7 other authors (1994). Studies of the conformation-dependent neutralizing epitopes of simian immunodeficiency virus envelope protein. J Virol 68, 26242631.[Abstract]
Kazlauskaite, J., Sanghera, N., Sylvester, I., Vénien-Bryan, C. & Pinheiro, T. J. T. (2003). Structural changes of the prion protein in lipid membranes leading to aggregation and fibrillization. Biochemistry 42, 32953304.[CrossRef][Medline]
Krachnarov, C. P., Kayman, S. C., Honnen, W. J., Trochev, O. & Pinter, A. (2001). V3-specific polyclonal antibodies affinity purified from sera of infected humans effectively neutralize primary isolates of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 17, 17371748.[CrossRef][Medline]
Labrijn, A. F., Poignard, P., Raja, A. & 16 other authors (2003). Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 77, 1055710565.
Labrosse, B., Treboute, C., Brelot, A. & Alizon, M. (2001). Cooperation of the V1/V2 and V3 domains of human immunodeficiency virus type 1 gp120 for interaction with the CXCR4 receptor. J Virol 75, 54575464.
Laune, D., Molina, F., Ferrieres, G. & 7 other authors (1997). Systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins. J Biol Chem 272, 3093730944.
Levi, M., Sällberg, M., Rudén, U., Herlyn, D., Maruyama, H., Wigzell, H., Marks, J. & Wahren, B. (1993). A complementarity-determining region synthetic peptide acts as a miniantibody and neutralizes human immunodeficiency virus type 1 in vitro. Proc Natl Acad Sci U S A 90, 43744378.
Levy, J. A. (1998). HIV and the Pathogenesis of AIDS, 2nd edn, pp. 359. Herndon, VA: American Society of Microbiology.
Losman, B., Bolmstedt, A., Schønning, K., Björndal, A., Westin, C., Fenyö, E. M. & Olofsson, S. (2001). Protection of neutralization epitopes in the V3 loop of oligomeric human immunodeficiency virus type 1 glycoprotein 120 by N-linked oligosaccharides in the V1 region. AIDS Res Hum Retroviruses 17, 10671076.[CrossRef][Medline]
Mbah, H. A., Burda, S., Gorny, M. K., Williams, C., Revesz, K., Zolla-Pazner, S. & Nyambi, P. N. (2001). Effect of soluble CD4 on exposure of epitopes on primary, intact, native human immunodeficiency virus type 1 virions of different genetic clades. J Virol 75, 77857788.
McLain, L. & Dimmock, N. J. (1994). Single- and multi-hit kinetics of immunoglobulin G neutralization of human immunodeficiency virus type 1 by monoclonal antibodies. J Gen Virol 75, 14571460.[Abstract]
Millar, A. L., Jackson, N. A. C., Dalton, H., Jennings, K. R., Levi, M., Wahren, B. & Dimmock, N. J. (1998). Rapid analysis of epitopeparatope interactions between HIV-1 and a 17-amino-acid neutralizing microantibody by electrospray ioniszation mass spectrometry. Eur J Biochem 258, 164169.[Abstract]
Minder, D., Böni, J., Schüpbach, J. & Gehring, H. (2002). Immunophilins and HIV-1 infection. Arch Virol 147, 15311542.[CrossRef][Medline]
Monnet, C., Laune, D., Laroche-Traineau, J. & 11 other authors (1999). Synthetic peptides derived from the variable regions of an anti-CD4 monoclonal antibody bind to CD4 and inhibit HIV-1 promoter activation in virus-infected cells. J Biol Chem 274, 37893796.
Moore, J. P., Thali, M., Jameson, B. A. & 12 other authors (1993). Immunochemical analysis of the gp120 surface glycoprotein of human immunodeficiency virus type 1: probing the structure of the C4 and V4 domains and the interaction of the C4 domain and the V3 loop. J Virol 67, 47854796.[Abstract]
Nehete, P. N., Vela, E. M., Hossain, M. M., Sarkar, A. K., Yahi, N., Fantini, J. & Sastry, K. J. (2002). A post-CD4-binding step involving interaction of the V3 region of viral gp120 with host cell surface glycosphingolipids is common to entry and infection by diverse HIV-1 strains. Antiviral Res 56, 233251.[CrossRef][Medline]
Parren, P. W. H. I. & Burton, D. R. (2001). The antiviral activity of antibodies in vitro and in vivo. Adv Immunol 77, 195262.[Medline]
Pinter, A., Honnen, W. J. & Tilley, S. A. (1993). Conformational changes affecting the V3 and CD4-binding domains of human immunodeficiency virus type 1 gp120 associated with Env processing and with binding of ligands to these sites. J Virol 67, 56925697.[Abstract]
Poignard, P., Saphire, E. O., Parren, P. W. H. I. & Burton, D. R. (2001). gp120: biologic aspects of structural features. Annu Rev Immunol 19, 253274.[CrossRef][Medline]
Pons, J., Rajpal, A. & Kirsch, J. F. (1999). Energetic analysis of an antigen/antibody interface: alanine scanning mutagenesis and double mutant cycles on the HyHEL10/lysozyme interaction. Protein Sci 8, 958968.[Abstract]
Rizzuto, C., Wyatt, R., Hernandez-Ramoz, N., Sun, Y., Kwong, P. D., Hendrickson, W. A. & Sodroski, J. (1998). A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 280, 19491953.
Saphire, E. O., Parren, P. W. H. I., Pantophlet, R. & 7 other authors (2001). Crystal structure of a neutralizing human IgG against HIV-1: a template for vaccine design. Science 293, 11551159.
Smith, J. W., Hu, D., Satterthwait, A. C., Pinz-Sweeney, S. & Barbas, C. F., III (1994). Building synthetic antibodies as adhesive ligands for integrins. J Biol Chem 269, 3278832795.
Thali, M., Furman, C., Wahren, B., Posner, M., Ho, D. D., Robinson, J. & Sodroski, J. (1992). Cooperativity of neutralizing antibodies directed against the V3 and CD4 binding regions of the human immunodeficiency virus gp120 envelope glycoprotein. J Acquir Immune Defic Syndr 5, 591599.[Medline]
Welling, G. W., Geurts, T., van Gorkum, J., Damhof, R. A., Drijfhout, J. W., Bloemhoff, W. & Welling-Wester, S. (1990). Synthetic antibody fragment as ligand in immunoaffinity chromatography. J Chromatogr 512, 337343.[CrossRef][Medline]
Welling, G. W., van Gorkum, J., Damhof, R. A., Drijfhout, J. W., Bloemhoff, W. & Welling-Wester, S. (1991). A ten-residue fragment of an antibody (mini-antibody) directed against lysozyme as ligand in immunoaffinity chromatography. J Chromatogr 548, 235242.[CrossRef][Medline]
Willey, R. L., Ross, E. K., Buckler-White, A. J., Theodore, T. S. & Martin, M. A. (1989). Functional interaction of constant and variable domains of human immunodeficiency virus type 1 gp120. J Virol 63, 35953600.[Medline]
Williams, W. V., Guy, H. R., Rubin, D., Robey, F., Myers, J. N., Kieber-Emmons, T., Weiner, D. B. & Greene, M. I. (1988). Sequences of the cell-attachment sites of reovirus type 3 and its anti-idiotypic/antireceptor antibody: modeling of their three-dimensional structures. Proc Natl Acad Sci U S A 85, 64886492.
Williams, W. V., London, S. D., Weiner, D. B., Wadsworth, S., Berzofsky, J. A., Robey, F., Rubin, D. H. & Greene, M. I. (1989a). Immune response to a molecularly defined internal image idiotope. J Immunol 142, 43924400.
Williams, W. V., Moss, D. A., Kieber-Emmons, T., Cohen, J. A., Myers, J. N., Weiner, D. B. & Greene, M. I. (1989b). Development of biologically active peptides based on antibody structure. Proc Natl Acad Sci U S A 86, 55375541.
Williams, W. V., Kieber-Emmons, T., VonFeldt, J., Greene, M. I. & Weiner, D. B. (1991a). Design of bioactive peptides based on antibody hypervariable region structures. Development of conformationally constrained and dimeric peptides with enhanced affinity. J Biol Chem 266, 51825190.
Williams, W. V., Kieber-Emmons, T., Weiner, D. B., Rubin, D. H. & Greene, M. I. (1991b). Contact residues and predicted structure of the reovirus type 3-receptor interaction. J Biol Chem 266, 92419250.
Winter, G. & Milstein, C. (1991). Man-made antibodies. Nature 349, 293299.[CrossRef][Medline]
Yang, W.-P., Green, K., Pinz-Sweeney, S., Briones, A. T., Burton, D. R. & Barbas, C. F., III (1995). CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J Mol Biol 254, 392403.[CrossRef][Medline]
Zhang, P. F., Bouma, P., Park, E. J., Margolick, J. B., Robinson, J. E., Zolla-Pazner, S., Flora, M. N. & Quinnan, G. V., Jr (2002). A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4-independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response. J Virol 76, 644655.
Zhang, M.-Y., Shu, Y., Rudolph, D., Prabakaran, P., Labrijn, A. F., Zwick, M. B., Lal, R. B. & Dimitrov, D. S. (2004). Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning. J Mol Biol 335, 209219.[CrossRef][Medline]
Zwick, M. B., Parren, P. W. H. I., Saphire, E. O., Church, S., Wang, M., Scott, J. K., Dawson, P. E., Wilson, I. A. & Burton, D. R. (2003). Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120. J Virol 77, 58635876.
Received 8 December 2004;
accepted 9 February 2005.
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