Analysis of a 17-amino acid residue, virus-neutralizing microantibody

Caroline J. Heap{dagger},{ddagger}, Yuqin Wang{dagger}, Teresa J. T. Pinheiro, Steven A. Reading, Keith R. Jennings and Nigel J. Dimmock

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

Correspondence
Nigel J. Dimmock
n.j.dimmock{at}warwick.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The antibody-binding site, through which an antibody binds to its epitope, is a complex structure formed by the folding together of six complementarity-determining regions (CDRs). However, certain peptides derived from CDR sequences retain antibody specificity and function; these are know as microantibodies (MicroAbs). For example, the F58 MicroAb is a 17 residue, cyclized peptide (CDLIYYDYEEDYYFDYC) derived from CDR-H3 of F58, an IgG1 specific for the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). Both MicroAb and IgG recognize the same epitope in the V3 loop and, despite its small size, the MicroAb neutralizes the infectivity of HIV-1 IIIB only 32-fold less efficiently on a molar basis. The advantage of MicroAbs is that their small size facilitates structure–function analysis. Here, the F58 MicroAb was investigated using alanine scanning, mass spectroscopy and surface plasmon resonance. Neutralization of infectious IIIB was generally more sensitive to alanine substitution than binding to soluble gp120. There appeared to be a division of function within the MicroAb, with some residues involved in antigen binding (alanine substitution of 11D, 12Y or 13Y abrogated both binding and neutralization), whereas others were concerned solely with neutralization (substitution of 3L, 8Y or 14F abrogated neutralization, but not binding). The MicroAb is predominantly {beta}-sheet and has strong conformational constraints that are probably essential for activity. The MicroAb and soluble gp120 formed a 1 : 1 complex, with an association rate that was threefold greater than that with IgG and a faster dissociation rate. Its equilibrium dissociation constant is 37·5-fold greater than that of IgG, in line with neutralization data. This study demonstrates how MicroAbs can make a useful contribution to the understanding of antigen–antibody interactions.

{dagger}These authors contributed equally to this work.

{ddagger}Present address: Health Protection Agency, Porton Down, Salisbury SP4 0JG, UK.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
An antibody binds to its cognate epitope through its paratope, a domain formed from the six complementarity-determining regions (CDRs) situated within the variable portions of the immunoglobulin light and heavy chains. Thus, a bivalently binding IgG can attach to antigen by the combined action of 12 CDRs. A microantibody (MicroAb) is the minimum recognition unit of an antibody and comprises a peptide derived from usually just one CDR. Its activity will depend on both its sequence and conformation. Some antiviral MicroAbs retain the virus binding and infectivity neutralizing activities of the parent antibody. The small size of MicroAbs should enable residues that are key to virus binding and neutralization activities to be easily identified. MicroAbs also have a potential advantage over IgG as therapeutic antivirals as they are less immunogenic and should be useful in situations where larger antibodies cannot easily penetrate.

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 gp120–gp41 oligomer on the surface of HIV-1 virions.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and virus.
The CD4+ human T-cell lines, C8166 and H9 (Centralized Facilities for AIDS Reagents, NIBSC), were grown at 37 °C in RPMI 1640 medium (BioWhittaker Europe) supplemented with 10 % (v/v) heat-inactivated fetal calf serum (FCS; Labtech International) and 2 mM glutamine (Sigma), with no antibiotics. HIV-1 IIIB was produced by co-cultivating persistently infected H9 cells with non-infected H9 cells. Medium was changed at 48 h to ensure a harvest of fresh virus at 72 h. Culture fluid was clarified by centrifugation and stored in liquid nitrogen.

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 ml–1; 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 ml–1 for 1 h at 37 °C and inoculated as above. The virus–antibody 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 guanidine–HCl, 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 MicroAb–gp120 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 µl–1 and 300 µl was injected at a flow rate of 10 µl min–1 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 (16·7–416·5 nM) was injected for 4 min at 30 µl min–1. 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 (16·7–167 nM) was injected for 4 min at 30 µl min–1, 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 cm–1 in the range 1000–4000 cm–1. 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 (1600–1700 cm–1) 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 cm–1. Band assignments were made according to Cabiaux et al. (1989).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Binding of alanine-substituted MicroAbs to gp120
All residues, apart from the terminal cysteines that are required for peptide circularization, were sequentially substituted with alanine and the ability of each alanine-substituted F58 MicroAb to inhibit binding of F58 IgG to monomeric gp120 from strain IIIB was determined. Fig. 1(a) shows examples of inhibition of F58 binding by substituted MicroAbs. Fig. 1(b) summarizes the data as relative binding (at 25 % inhibition of IgG binding) with respect to the non-substituted MicroAb. Alanine substitutions of 11D, 12Y and 13Y virtually abolished relative binding (<18–26 %), suggesting that these are key residues for interaction with the epitope in the gp120 monomer. Three of 14 substitutions (at 5Y, 6Y and 9E) reduced relative binding of the MicroAb to 29–39 % and eight substitutions (at 2D, 3L, 4I, 7D, 8Y, 10E, 14F and 15D) gave relative binding of 51–115 %. Inhibition of IgG binding at 50 % was not achieved with peptides that were alanine-substituted at 11D, 12Y and 13Y (data not shown). No substitution increased the affinity of the MicroAb for its epitope.



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Fig. 1. The ability of alanine-substituted F58 MicroAbs to inhibit the binding of F58 IgG to solid phase soluble gp120. gp120 was incubated with a range of concentrations of MicroAb overnight at 20 °C. F58 IgG (0·01 µg per well) was then added for 1 h and its binding was detected by ELISA. (a) Sample curves calculated by Prism software ({blacksquare}, wt non-substituted MicroAb; {blacktriangleup}, MicroAb 5YA; {blacktriangledown}, MicroAb Y13A). (b) Relative binding of the MicroAbs, which is calculated from the concentration of MicroAb required to inhibit the binding of F58 IgG by 25 %. Data are normalized to those of wt MicroAb. {downarrow} indicates that MicroAb12 inhibited F58 binding by <25 % at 100 µg ml–1 and has a relative binding value of <18 %. Data are the mean of at least two independent experiments. Insufficient 16Y-A MicroAb was produced to be used here. Error bar, standard error of the mean.

 
Neutralization of infectious HIV-1 by alanine-substituted MicroAbs
Fig. 2(a) shows examples of neutralization curves. Data are summarized in Fig. 2(b) as neutralization relative to wt MicroAb. The greatest reduction in relative neutralization (to <=4 % of wt) occurred when residues 9E, 11D, 12Y, 13Y and 14F were sequentially substituted by alanine. Substitution of residues 3L, 5Y and 6Y also severely compromised neutralization. Substituting residues 2D, 7D, 10E and 15D reduced relative neutralization to 25–37 %. Overall, neutralization by MicroAbs was more sensitive to alanine substitution than binding to gp120. Loss of neutralization correlated closely with loss of binding for residues 12Y and 13Y and, possibly, 11D. However, substitution 14F-A had little effect on binding, but virtually abolished neutralization (see Discussion).



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Fig. 2. Neutralization of HIV-1 IIIB by alanine-substituted F58 MicroAbs. Virus incubated with each MicroAb for 1 h at 37 °C was inoculated on to C8166 cells to determine the reduction in virus infectivity. (a) Sample curves showing neutralization activity ({blacksquare}, wt non-substituted MicroAb; {blacklozenge}, MicroAb D7A; {blacktriangledown}, MicroAb Y12A; {blacktriangleup}, MicroAb F14A). Data are the mean of two independent experiments, each with six replicate wells. Standard error of the mean for each data point is shown. (b) Relative neutralization. The concentration of MicroAb required for 75 % neutralization was calculated from the mean data in (a) and expressed relative to the neutralization achieved by the wt MicroAb (100 %). As relative neutralization is derived from the data means, it is not appropriate to calculate an error. {downarrow} indicates a MicroAb that neutralized by <75 % at the highest concentration used (30 µg ml–1) and has a relative neutralization value of <4 %.

 
Deuteration of MicroAb F58 and the V3 peptides Z321 and BRA and their analysis by MS
The MicroAb and peptides Z321 and BRA, which are derived from the V3 loop of the gp120 envelope glycoprotein of an A and a B clade primary virus isolate, respectively (see Methods), were deuterated and analysed. After dilution into D2O for 10 min, the MicroAb showed a mass increase over the aqueous form equivalent to an exchange of 20·5 of 31 (67 %) of its potentially exchangeable hydrogen moieties (Fig. 3a, b). No further exchange took place, even after 24 h incubation in D2O (data not shown). The MicroAb thus appears to have a substantial amount of conformational structure that prevents 33 % of its hydrogens from interacting with its aqueous environment. In contrast, 29 of 32 (91 %) of the potentially exchangeable hydrogens of the V3 loop peptide Z231 were deuterated (Fig. 3c, d) and 64 of 70 (92 %) of the potentially exchangeable hydrogens of peptide BRA were deuterated (Fig. 3e, f). Taking into account the possibility of back exchange, data suggest that both V3 peptides are effectively fully exchanged and hence unstructured, although the BRA peptide (like the MicroAb) is cyclized through a terminal disulfide bond.



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Fig. 3. MS of the F58 MicroAb (a, b), the HIV-1 V3 peptides Z321 (c, d) and BRA (e, f) showing the mass increase after deuteration. (a), (c) and (e) are in water and (b), (d) and (f) were analysed after deuteration in D2O for 10 min.

 
In order to determine if failure of complete H/D exchange in the MicroAb resulted from the structure adopted when the peptide was cyclized by disulfide bonding between the terminal cysteine residues, the experiment was repeated after reduction and alkylation in the presence of 6 M guanidine–HCl. After desalting, there was 63 % H/D exchange, compared with 67 % in the unmodified peptide. This corresponded to 22 of the 35 potentially exchangeable hydrogens and suggested that the peptide conformation was essentially unaltered by loss of the disulfide linkage (data not shown). To further test the stability of its structure, denatured and alkylated MicroAb was buffer-exchanged, refolded in the absence of denaturant and then incubated in D2O at 100 °C for 1 h. The H/D exchange was not demonstrably different from the control, untreated MicroAb indicating that its conformation was essentially unchanged (data not shown). It appears, therefore, that the disulfide bond is not required for MicroAb folding, although it may stabilize conformation. Biological activity of the reduced MicroAb was confirmed by its ability to form complexes with the V3 peptides indistinguishable from those formed with cyclized MicroAb (see below).

Analysis of deuterated MicroAb–antigen complexes by MS
MicroAb–V3 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 MicroAb–Z321 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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Fig. 4. Analysis of MicroAb–Z321 V3 peptide complexes. (a) The complex formed with aqueous MicroAb and Z321 V3 peptide. (b) Analysis of the aqueous MicroAb–Z321 V3 peptide complex after incubation in D2O for 10 min. (c) The complex formed from pre-deuterated reactants. All complexes were 1 : 1.

 


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Fig. 5. Rate of H/D exchange of deuterated MicroAb ({bullet}) or deuterated MicroAb–Z321 V3 peptide complexes ({blacklozenge}). Data are representative of two independent experiments.

 
Comparison of the affinity of F58 MicroAb and F58 IgG for gp120
Biotinylated MicroAb was immobilized onto the surface of a streptavidin-coated chip and F58 IgG was immobilized by amine chemistry on a CM5 chip, preparatory to monitoring gp120 capture by surface plasmon resonance. Kinetic constants calculated from the sensorgrams are shown in Table 1. The Ka of the MicroAb was approximately threefold higher than that of the IgG, as expected from the greater mobility associated with a smaller molecule. However, its dissociation rate (Kd) was 100-fold greater. Both equilibrium values (KD) were high: IgG was 0·2 nM and MicroAb was 7·5 nM. Thus, IgG had a 37·5-fold higher affinity than the MicroAb.


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Table 1. Kinetics of F58 MicroAb–gp120 and F58 IgG1–gp120 interactions as measured by SPR

 
Structural analysis of F58 MicroAb and the V3 peptides Z321 and BRA by ATR FTIR spectroscopy
Spectra in Fig. 6 show amide I, II and II' bands at 1700–1600 cm–1, 1600–1500 cm–1 and 1500–1400 cm–1, respectively, which arise from the carbonyl stretching and NH bending and stretching vibrations of the peptide groups. Different bands under the amide I envelope reflect the various types of secondary structure. F58 MicroAb and Z321 have a strong band at 1636 cm–1 and 1621 cm–1, respectively, indicating a high content of {beta}-sheet structure (Fig. 6a, c), whereas BRA has a strong component under the amide I band at around 1647 cm–1, which can be attributed to {alpha}-helical structure (Fig. 6b). BRA and Z231 also show a strong band around 1674 cm–1, indicating the presence of {beta}-turns (Fig. 6b,c). The sharp band at 1514 cm–1 in the spectrum of the MicroAb arises from tyrosine absorbance, in accordance with its high tyrosine content (6 of 17 residues). From analysis of the intensity of the various spectral components under the amide I, it was concluded that F58 is 50–60 % {beta}-sheet, Z231 is over 30 % {beta}-sheet and BRA has 20–30 % {alpha}-helix and random coil. A short pulse of H/D exchange in FTIR experiments aids the separation of {alpha}-helical structure from random coil and provides information on solvent accessibility, hydrogen bonding and general structural stability. In an FTIR spectrum, the extent of H/D exchange on the peptide amide group can be evaluated by the decay in intensity of the amide II as it shifts by 100 cm–1 towards lower wavenumbers, giving rise to the so-called amide II' band. F58 MicroAb had the highest intensity associated with the amide II band at 1570 cm–1 after H/D exchange, whereas BRA and Z231 had little intensity in this region (Fig. 6); thus, the MicroAb is more stably folded.



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Fig. 6. ATR FTIR spectra of (a) F58 MicroAb, (b) V3 peptide BRA and (c) V3 peptide Z231 after H/D exchange for 10 min.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The analysis of FAbs or single chain variable fragments by amino acid residue substitution is a laborious exercise, as random or defined mutations have to be inserted using molecular cloning with techniques such as PCR or alanine scanning mutagenesis (e.g. Yang et al., 1995; Pons et al., 1999; Calarese et al., 2003; Zwick et al., 2003; Zhang et al., 2004). However, it has been shown in this report that such analysis can be readily undertaken with MicroAbs as their small size allows variant molecules to be synthesized chemically.

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 virion–cell 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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Table 2. Relative binding and neutralization of HIV-1 by alanine-substituted MicroAbs

Data are taken from Figs 1 and 2. See Methods and figure legends for determination of relative values. ND, Not done; NA, not applicable.

 
Data above show that the F58 MicroAb is strongly conformational, with only 67 % of its available hydrogens able to exchange with deuterium. Denaturation, reduction and alkylation of the cyclized MicroAb did not affect its structure or biological activity suggesting that the disulfide linkage was not essential for its conformation. This might appear at first sight to contradict earlier data showing that the cyclized MicroAb bound to soluble gp160 and neutralized infectious virus better than its linear form (Levi et al., 1993), but the latter was synthesized as a linear molecule, whereas in this study, a cyclized peptide was reduced and alkylated. It may be that despite reduction and alkylation, the cyclic peptide retains sufficient internal bonding to be able to refold into something like its native conformation. These data are consistent with FTIR spectra, which show a predominantly {beta}-sheet, stable structure for F58 MicroAb.

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 virus–MicroAb 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 MicroAb–V3 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 virion–cell 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 % {beta}-sheet and BRA being 20–30 % {alpha}-helix and random coil. Both have a high {beta}-turn content (Fig. 6b, c: band at 1674 cm–1). 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.


   ACKNOWLEDGEMENTS
 
We thank the Biotechnology and Biological Sciences Research Council, UK, for financial support and the Medical Research Council, UK, for a studentship (C. J. H.). BIAcore 2000 was purchased with the help of a grant from the Joint Research Equipment Initiative, UK. We are grateful to Lesley McLain for excellent technical assistance, Iman Bath for collecting FTIR spectra, Britta Wahren (Swedish Institute for Infectious Disease Control, Solna, Sweden) for mAb F58, and the Centralized Facilities for AIDS Reagents, NIBSC, Potters Bar, UK, and the NIH AIDS Research and Reference Reagent Program for reagents.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 8 December 2004; accepted 9 February 2005.



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