Constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-HIV-1 mAb 2F5

Immunology Letters 136 (2011) 80–89

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Immunology Letters journal homepage: www.elsevier.com/locate/immlet

Constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-HIV-1 mAb 2F5 Yadira Palacios-Rodríguez a , Tatiana Gazarian b , Leonor Huerta c , Karlen Gazarian a,∗ a

Department of Molecular Biology and Biotechnology, Institute of Biomedical Research, Mexican National Autonomous University, Circuito Exterior, Mexico City 04510, Mexico Department of Public Health, Faculty of Medicine, Mexican National Autonomous University, Ciudad Universitaria, 04510 Mexico City, Mexico c Department of Immunology, Institute of Biomedical Research, Mexican National Autonomous University, Circuito Exterior, Mexico City 04510, Mexico b

a r t i c l e

i n f o

Article history: Received 18 August 2010 Received in revised form 2 December 2010 Accepted 26 December 2010 Available online 13 January 2011 Keywords: HIV-1 mAb 2F5 Phage display Mimotope Epitope

a b s t r a c t The monoclonal antibody 2F5 (mAb 2F5), one of the most potent broadly neutralizing mAbs targeted to the HIV-1 gp41 membrane proximal exterior region (MPER), displays an unusually wide antigenic specificity, tolerating amino acid substitutions at virtually all positions of the 662-ELDKWAS-668 epitope sequence when presented by peptides. Investigating this phenomenon, Menendez et al. [22] concluded that the paratope of 2F5 contains two distinct binding compartments. One is specific and binds the DKW epitope core; the other is multi-specific and binds to the flanking DKW regions that can be distinct from the epitope sequence. Because the DKW-flanking amino acids are strongly conserved in viruses, it is not clear whether the DKW only satisfies the 2F5 epitope recognition demand. In this study, we demonstrate that the specificity of recognition of the epitope depends on the structural context in which the cognate epitope sequence is presented. The antibody does not tolerate any replacements of the DKW-flanking epitope amino acids and binds exclusively to the (L)DKWA sequence provided that it is presented by a 7-mer constrained peptide exposed by the M13 phage pIII protein. Our data propose a novel epitope recognition model in which the 2F5 mAb requires a sequence longer than DKW and no substitution of flanking amino acids for specific recognition of the peptide. Additionally, immunization data supports the notion that the binding and neutralizing immunogenic structural features of the described epitope model do not coincide. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The monoclonal antibody 2F5 (mAb 2F5) isolated in the early ‘90s [1], is a potent human anti-HIV-1 neutralization antibody [2–8] targeted to the gp41 glycoprotein membrane proximal exterior region (MPER). Since it was isolated, the 2F5 mAb and its epitope have continued to be the focus of extensive investigations attempting to elucidate the mechanism by which mAb 2F5 impedes viral entry into host cells of many HIV-1 clades. Screening phage display peptide libraries with 2F5 [9,10] examining binding to synthetic peptides [11] demonstrated that the mAb 2F5 recognized sequence motifs corresponding to the HIV-1 gp41 662 ELDKWAS668 site as the epitope core. Crystallographic data highlighted the 664 DKW666 ␤-turn motif and side chain contacts as critical for the binding of

∗ Corresponding author at: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, Ciudad Universitaria, Circuito escolar s/n, C.P. 04510, Mexico, D.F., Mexico. Tel.: +52 55 5622 9206/9207; fax: +52 55 5622 9212. E-mail address: [email protected] (K. Gazarian). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2010.12.008

2F5 [12–16]. In line with this in vitro mapping data, DKW was the minimal structure necessary for neutralization in pseudoviruses [17]. In the gp41 protein-antibody complex, a longer sequence encompassing the ELDKWAS was protected from proteolysis [18]. Consistent with this, the elongation of peptides beyond the ELDKWAS sequence [19], enhancement of its alpha-helicity in the context of DP178 [20] and constraint posed by means of cysteines via disulfide-bridged loop formation or by incorporation of a sidechain to side-chain lactam bridge [19,21,22] enhanced 2F5 binding affinity to the epitope. In those assays, the antibody consistently showed tolerance to substitution of the ELDKWAS amino acids, except the aspartic acid and the tryptophan. This contradicted conservation of the ELDKWAS sequence in the B-subtype viruses. More recently, Menendez et al. [22] screened a panel of 17 libraries of linear and constrained peptides displayed by the pVIII major phage protein against mAb 2F5 and assessed in detail the roles of critical and replaceable parts of the ELDKWAS sequence. The results of this comprehensive analysis revealed that the paratope of 2F5 consists of two binding compartments: one specific compartment that recognizes the DKW (or DRW, as the lysine is replaceable by arginine) and a “multi-specific” compartment that interacts with

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the epitope regions surrounding this core in a non-specific fashion (i.e., tolerating amino acid differences from the MPER sequence). It is remarkable that this is also true for the amino acids that are necessary for virus neutralization by 2F5 [17]. Whether these distinct binding functions are structurally separated in the paratope or whether the same paratope region displays them in a structurally different state adopted under the influence of co-factors is yet to be investigated. ELDKWAS-containing peptides inserted into different carriers used as immunogens did not induce antibodies with the 2F5 neutralizing potential [23–26]. An explanation of this inability is that a viral membrane component is a part of the 2F5 epitope and that the paratope interacts apart from the MPER ELDKWAS with the membrane via its long CDR H3 loop crown (see recent study by Ofek et al. for more information Ref. [27]). In this study, we show that when mAb 2F5 screens a pIII-type phage display 7-mer constrained peptide library for its epitope mimics (not screened in previous experiments with 2F5 [9,10,22]), it demands an epitope sequence longer than DKW and does not tolerate substitutions in the epitope amino acid sequence. 2. Materials and methods 2.1. Synthetic peptides, mAb 2F5, anti-CD4 mAb, cells The synthetic peptides with overlapping 15-mer gp41 MPER sequences encompassing the ELDKWAS sequence, the mAb 2F5, the anti-CD4 mAb and the Jurkat cell lines E6-1 and HXBc2 which are stably transfected with the env (envelope) gene from the HIV-1 HXBc2 strain were from NIH AIDS Research and Reference Reagent Program, USA. 2.2. Peptide libraries The 12-mer linear and 7-mer cysteine-constrained libraries were purchased from New England BioLabs Inc. (Beverly, MA, USA). In each of them, random peptides (approx. 2.7 × 109 electroporated sequences) are fused to a minor coat protein (pIII) of the M13 phage and expressed at its N-terminus separated by a Gly–Gly–Gly–Ser spacer. Shortly before use, libraries were amplified by the manufacturer to contain approximately 20 copies of each sequence per 10 ␮l, which is the amount used in a single biopanning experiment. 2.3. Biopanning and characterization of selected peptides The biopanning procedure was essentially as described by Smith and Scott [28] with adjustments [29]. Briefly, two wells in a 96well polystyrene microtiter plate (Immulon 4 flat bottom plates, Dynatech Lab Inc., USA) were coated with mAb 2F5. For these experiments, one well was filled with 150 ␮g/ml mAb in PBS and the second with 75 ␮g/ml of mAb, each in 100 ␮l PBS. The plate was kept overnight at 4 ◦ C with gentle rocking. Unbound mAb was discarded, and the wells were washed 6 times with PBS-T (PBS-0.1% Tween 20) and blocked for 1 h at 4 ◦ C with blocking buffer (PBS1% BSA), followed by five consecutive washing steps using PBS-T. For affinity selection, 10 ␮l of the library (2 × 1010 plaque-forming units, p.f.u.) was added to 190 ␮l of PBS-T, and the mixture was distributed in the two wells (100 ␮l per well) with immobilized and blocked 2F5. Wells were then incubated for 1 h at RT while rocking gently to allow phages to bind. The unbound phages were pipetted out, and wells were washed 10 times with PBS-T at RT. Bound phage was eluted from each well by stirring with 100 ␮l of elution buffer (0.1 N HCl–glycine, pH 2.2). Eluted phage (designated further as “eluate”) from two wells was combined and quickly neutralized by addition of 35 ␮l of 2 M Tris base. Phage was amplified by infection of lag-phase E. coli ER2738 (BioLabs) on LB plates, and the num-

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ber of plaques was quantified to estimate the phage titer (p.f.u.) Approximately 70% of the eluate was amplified in 30 ml of 2x YT for 4 h. After pelleting the cells by centrifugation, the supernatant was collected, and the phage was precipitated by adding PEG/NaCl (20%/40%) at 4 ◦ C. Precipitated phage was pelleted by centrifugation (11,000 rpm for 10 min at 4 ◦ C), re-suspended in PBS and clarified by centrifugation (11,000 rpm for 2 min at 4 ◦ C). The supernatant was transferred to a fresh centrifuge tube for repeated phage precipitation with PEG/NaCl as described. The recovered phage titers were examined as indicated above, re-suspended in PBS at concentrations of 1012 –1013 p.f.u per ml and used in the second and third rounds of biopanning as described. In the third round of panning, the stringency conditions for washing were increased by using PBS0.5%-Tween-20 (instead of the 0.1% used in the first two rounds), and the eluted phage was not amplified and stored for short periods at 4 ◦ C and for longer periods in 50% glycerol at −35 ◦ C. Sequences and reactivities of selected phage-peptides were determined by cloning an aliquot of the eluate containing approximately 100 p.f.u. from each round to infect the E. coli ER 2737 strain. Cells were plated in a Luria agar dish with X-gal and IPTG. After overnight growth at 37 ◦ C, individual plaques were randomly picked and amplified in 3 ml of 2xYT medium as described for the amplification of eluates in the 2nd and 3rd rounds of biopanning. Amplified single-plaque phage (1012 –1013 p.f.u.) representing individual clones were used for further analysis. Two small samples were taken from each clone, one for purification of single-strand (ss) phage genomic DNA, and the second for assessment of reactivity with selecting IgG. Using ssDNA isolated from each clone, we sequenced the 5 region of the pIII gene with the inserted oligonucleotides using S35 labeled di-deoxy-nucleotides, primer (5 -CCAGACGTTAGTAAATG3 ) and T7 sequenase (Amersham, Life Science, Cleveland, OH, USA). The amino acids were deduced from the nucleotide codons to obtain the sequence of the selected peptide. 2.4. Mouse and rabbit immunization with selected peptides and serum testing by ELISA and in cell-cell fusion system Female 3- to 4-week-old C57BL/6 mice and 2.5 kg New Zealand rabbits were immunized as described in [30]. Briefly, 1011 –1012 p.f.u. phages per 20 g body weight were injected three times in 15-day intervals intraperitoneally to mice and intradermally (in three sites) to rabbits. Twelve days after the last injection, blood was collected from ear vein (rabbit) and by heart puncture of anaesthetized animals. Experiments with animals were performed in accordance with the Mexican official standard NOM062-ZOO-1999 (Specific techniques for the production, care, and use of laboratory animals). Sera prepared prior to (pre-immune) and after (immune) immunization were stored in aliquots at −20 ◦ C until they were used. The IgG fraction was affinity purified by mixing sera with Protein GSepharose gel in tubes and centrifuging and eluting the bound Ig (see for a more details [29]). For reactivity assessment, synthetic peptides at 5 ␮g/ml were prepared in 0.2 M pH 9.5 carbonate buffer to coat 96-well plates by overnight incubation at 4 ◦ C followed by washing steps with PBST 0.1%. The wells were blocked with PBS-3%BSA (300 ␮l/1 h/37 ◦ C) and the corresponding Ab was added (2F5 was diluted at final concentration of 0.1 ␮g/ml in PBST 0.2%-BSA 0.2%, in case of serum or IgG of immunized animals the amount or dilution is indicated in figures) at final volume of 100 ␮l during 1 h at 4 ◦ C. Bound Ab was detected using the corresponding secondary Ab AP conjugate (Zymed Laboratories Inc., USA) diluted 1:1000. The reaction was developed with p-nitrophenil phosphate substrate diluted in diethanolamine buffer at 37 ◦ C, absorbance was read at 405 nm in automated reader.

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2.5. Cell culture and cell–cell fusion assays Cell lines were cultured in RPMI supplemented with 10% FBS (Gibco BRL), 50 U/ml penicillin and 50 U/ml streptomycin (RPMI10) at 37 ◦ C with 5% CO2 . The fusogenic HXBc2 cell line was grown in the presence of 1 ␮g/ml tetracycline and selection antibiotics (200 ␮g/ml G418 and 200 ␮g/ml hygromycin) as described previously [31–36]. Expression of the gp120/gp41 viral env protein was induced by removing tetracycline by washing the cells with PBS and incubating in RPMI-10 with selection antibiotics for 3 days. The cell–cell fusion protocol developed by Huerta et al. [31] and subsequently extensively employed by Huerta et al. [32], López-Balderas et al. [33], Huerta et al. [34], Rivera-Toledo et al. [35] was used in these experiments to test the anti-mimotope serum antibodies. The protocol involves labeling fusion partner cells with the fluorescent carbocyanines DiI and DiO (Molecular Probes). Jurkat cells stably transfected with a plasmid bearing the env gene from the HIV-1 syncytium-inducing virus HXBc2 (DIOHXBc2 cells) and untransfected DiI-E6 labeled Jurkat cells as the CD4+ fusion partner [36] were co-cultured for 5 h in serum-free AIM-V medium (Invitrogen), and the fusion was determined as percentage of dual-fluorescent cells. The dependence on the interaction with CD4 was tested by the addition of an anti-CD4 mAb (clone RPA-T4; BD Pharmingen). To evaluate the inhibition effect, the Env+ cells (2 × 105 ) were pre-incubated with 2F5, IgG fraction or serum for 40 min at 37 ◦ C in 5% CO2 , after which the CD4+ E6 cells (2 × 105 ) were added. The syncytium formation was induced by co-culturing for 5 h, followed by collection and washing with 1x PBS and finally resuspension in FACS buffer. The analysis was performed by acquiring 10,000 events in a FACScalibur flow cytometer (Becton–Dickinson, San Jose, CA). The samples were gently pipetted just before FACS analysis to dissociate cell aggregates. 2.6. Statistical analysis Statistic analysis was done using the GraphPad Prism Software Inc. p-values < 0.05 were considered as statistically significant. 3. Results 3.1. Peptides from the 12-mer linear library The collection of peptides selected from the library of linear 12mers was composed of 53 sequenced peptides with the ELDKWAS motif. Of these, 45 were unique sequences with either the DKW or DRW (in 20%) amino acids that are indispensible for binding (Fig. 1A). The peptide sequences contained various substitutions at positions EL and AS flanking the DKW or DRW core motif (DPW in one clone, Fig. 1A). In general, this selection reproduced the pattern of ELDKWA-related motif sequences observed in the earlier selections with 2F5 [22] that amply exhibited the tolerance of mAb 2F5 for amino acid variations at all but two ELDKWAS positions: D and W. Meanwhile there was a noticeable tendency for preferential selection of peptides containing sequences longer than DKW. To yield additional information on these antibody preferences, we followed the quantitative changes in the screened library of different length sequences, i.e., DKW, DKWA, DKWAS, LDKWA, that resulted from biopanning. These reflected the importance of the DKW-flanking residues for recognition by 2F5 (Table 1). Their frequency in the sample relative to the estimated probability to occur in the library prior to selection (calculated using the equation provided by the Biolabs Inc., USA) was indicative of the contribution of residues outside DKW to the selection process. This was taken as an indicator of the selection efficiency, referred to as “enrichment” (Table 1). Using this as a criterion, it was not DKW alone,

but longer sequences including DKWA and LDKWA, that preferentially accumulated in the library (see Fig. 2 for visual effect). Formally, the size of the analyzed portion of the library was far from being representative. However, repeated biopanning and amplification greatly reduces the diversity of the library such that the most efficiently selected species form a majority of the population. Sequencing 40–50 random clones is usually sufficient for drawing conclusions about the principal antibody epitope priorities. With these reservations in mind, the DKWA and LDKWA sequences were more attractive for the antibody than DKW, which was amply presented in the library (Table 1). 3.2. Peptides from the constrained 7-mer library The use of the constrained 7-mer library strengthened the revelation of the antibody’s preference by restricting the selection to the two longer sequences. After 3 rounds, 74% of sequenced peptides were carriers of the epitope sequence. All 27 sequences displayed by 42 clones (some in copies) exclusively presented these two longer sequences (Fig. 1B, Table 1B), none of which contained only DKW. Thus, the constrained form of peptides, known to enhance the affinity of the binding [19,21,22,24], led to a lack of DKW alone or peptides with substitutions at various positions, including the arginine to lysine substitutes to give DRW; all these were selected from the 12-mer linear peptide library (Fig. 1A, Table 1A). In this selection, DKWA and LDKWA were the only adequate epitope models. The other restriction concerns the position of these sequences in this 7-mer cyclic peptide (peptides in cys–cys-constrained libraries are potent to form cyclic structure [22]); from the two selected sequence types, C-DKWAxxx-C and CxxLDKWA-C, it is evident that the structural context of this peptide (unlike the linear peptide) strongly determined the amino acid positions destined for the epitope. The DKWA sequence was exclusively at N-terminal positions 1–4, and the LDKWA was at C-terminal positions 3–7, leaving the rest of the positions free of the epitope (marked as “x”). The lack of selected peptides containing the ELDKWAS residues “E” and “S” to occupy these free positions thus extending the epitope presentation by the peptide might be due to various reasons. These epitope amino acids may not be critical for binding, they may have spatial hindrance at these loop sites, or these positions may be required for some “supporting” amino acids that are usually required for optimal spatial exposition of epitope mimics displayed by phage. Two points in this result are worth emphasizing. First, there was a strong discrimination in the selection of DKW-containing peptides as well as peptides with longer gp41 sequence but lacking the cognate epitope residues. In this structural context, neither lysine in the DKW nor amino acids flanking it were permissible, although they were allowed in complexes formed by the whole 2F5 molecule with many other types of peptides in biochemical experiments and by the 2F5 Fab’ fragment in crystal complexes [16]. 3.3. Immunogenicity of selected peptides As noted in Section 1, the use of ELDKWAS peptides as immunogens has not yet resulted in the induction of a 2F5-related neutralizing antibody. This is explained (but not yet proved) by the lack in immunogenic formulations of all viral structures that interact with 2F5, such as the membrane which is considered to be a constituent of the complete epitope ([13], reviewed [37]). In those experiments the peptides were presented in various structural contexts, for example, in variable loops of the HIV-1 gp120 [25] or in permissive sites of the MalE protein [26] or other scaffolds [38]. Because peptides selected by mAb 2F5 from phage libraries were not investigated thoroughly as immunogens, we decided to check whether our peptides-on-phage will be better immunogens. We therefore performed immunization of mice and

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83

Sequences of peptides selected from 12mer liner (A) and 7mer constrained (B) libraries (B) 7mer constrained*

(A) 12mer linear* EHY

LDKWAS HDM

LPQLNT ASS AAMTNHV YK YPT

LDKWA LDKWA LDKWA LDKWA LDKWA

T2 LYTS 2

KHLA LPH ITPA QVP MD S NGLDIS HPLTL

LDKWS LDKWS LDKWS LDKWS LDKWN LDKWV LDKWG LDKWT

I LA SITP TRF VAWP MKSLT LAPLAY V LL

QSH ALS YGFP

PSLSK MWMH

YDKWAS WGF EDKWAS TAS YDKWAS RP

N YL ATN EHA T QFI AIQLV HLPYT Y

MDKWA PDKWA Y DKWA YDKWA PDKWA PDKWA Y DKWA SDKWA MDKWA

AVFQSK 2 TPQMI LPYT QRXX LPHPTL RSPN MP LM NIVALR

SHPG T T TNL YPN AND SPI TPF YLP SWP

PDKWQ PDKWY PDKWS FDKWS I DKWV PDKWN HDKWS LDKWV TDKWS FDKWS

TLP2 GLAPYR2 YLSNLQ YLAS ALYH LTPL DLSR ALKP HLRT QSLS

SLHETHM TTLQT LESS QT LV MPD ALV ETLA

LDRWA LDRWS QL2 LDRWT TLA 2 MDRWAS LRWS PDRWA FLQMS2 PDRWA LWPL S DRWS WMHQ KDRWV TLG DPWA FRWPSGPM Summary: N=53, 45 sequences E=0, L=40, D=100, K=77/R=21, W=100, A=51, S=9 *superscript number: frequency of selection

KT MA WH WT LV LR VS LG

LDKWA 4 LDKWA 2 LDKWA 2 LDKWA LDKWA LDKWA LDKWA NDKWA

DKWA PPT5 DKWA GLL4 DKWA PTS3 DKWA LAY 2 DKWA PRS DKWA VRW DKWA PPS DKWA EQY DKWA SQP DKWA FMT DKWA PPG DKWA PSA DKWA PSW DKWA QTY DKWA TRF DKWA GRA DKWA GPR DKWA PRQ DKWA IPW Summary: N=42 clones, 27 sequences, E=0, L=29, D=100, K=100, W=100,A=100,S=0 *superscript number: frequency of selection

2F5

L

D

W

K

A

X

X

W

K

X

X

A

D

X

C-C G G G S

linker pIII

g

pphage

C-C G G G S

linker pIII

phage

p

(C) 7mer cyclic peptide presenting the epitope DKWA on the N-terminal and LDKWA on C -terminal ends.

Fig. 1. Sequences found in the 100% (A) and 74% (B) of analyzed clones from the 3-rd round eluates in biopanning experiments with 12-mer linear and 7-mer constrained libraries, respectively. (C) Representation of DKWA and LDKWA sequences in the putative phage-displayed loop.

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Table 1 Statistical data on contribution of gp41 MPER residues to the selection by mAb2F5 of different-length and sequence epitope versions from the 12mer linear and 7mer constrained libraries. Epitope motifa

In the initial library

A. 12mer library LDKWAS LDKWA LDKW DKWAS DKWA DKW LDRW LDRWA DRWA DRW DPWA

In selected library

Probabilityb (p)

pa complexity of the libraryc

%P

Frequency

6.74 × 108 − 7.70E−07 1.44E−05 8.30E−07 9.31E−06 1.72E−04 2.42E−05 1.29E−06 1.60E−05 2.90E−04 4.10E−05

1.82E+02 2.08E+03 3.89E+04 2.24E+03 2.51E+04 4.64E+05 6.53E+04 3.48E+03 4.32E+04 7.83E+05 1.11E+05

6.74E−06 7.70E−05 1.44E−03 8.30E−05 9.31E−04 1.72E−02 2.42E−03 1.29E−04 1.60E−03 2.90E−02 4.10E−03

1 7 8 3 10 12 4 1 4 2 1

Total

1.37E+01 6.65E+02 9.23E+03 7.52E+02 1.37E+04 1.78E+05

1.14E−06 5.54E−05 7.69E−04 6.27E−05 1.14E−03 1.48E−02

c

– 28.6 71.4 – – –

42

– 5.16E+05 9.29E+04 – – –

100

The ELDKWAS sequence is not included because it was not found among sequenced peptides. Calculated using the equation of the library manufacturer (Biolabs Inc., USA). The complexity of the library was 2.7 × 109 for 12mer linear library and 1.2 × 109 for 7mer constrained library.

rabbits with phage-displayed 12-mer or 7-mer peptides. Both mice and rabbits developed antibody reactivity to the synthetic peptide NEQELLELDKWASLW (Fig. 3A and B) representing the gp41 epitope, thus demonstrating that the phage-peptides displayed properties described in previous experiments with synthetic peptides [23]. The 7-mer constrained mimotope induced significantly

20.00

A.

3.500

1:100

A.

1:300

***

3.000

D.O. 405 nm

b

2.80E+05 1.72E+05 1.05E+04 6.82E+04 2.03E+04 1.32E+03 3.12E+03 1.46E+04 4.72E+03 1.30E+02 4.60E+02

100

0 12 30 0 0 0

Total

Enrichment

1.9 13.2 15.1 5.7 18.9 22.6 7.5 1.9 7.5 3.8 1.9

53

B. 7mer constrained library ELDKWA 1.14E−08 LDKWA 5.54E−07 DKWA 7.69E−06 LDRWA 6.27E−07 LDKW 1.14E−05 DKW 1.48E−04

a

%

2.500 2.000 1.500 1.000 0.500 0.000 1

10.00

3.500 5.00

3.000

O.D.405nm

Selected pepdes (x104)

15.00

0.00 LDKWA

15

DKWAS

DKWA

DKW

B.

2

B.

1:50

***

1:100

4

5

6

1:300

***

***

2.500 2.000 1.500 1.000 0.500

Fold difference in selecon

3

#

#

#

0.000 10

1

5

0 LDKWA

LDRWA

DKW

DRW

DKWA

DRWA

Fig. 2. Graphical representation of Table 1 data on enrichment in selected 12-mer linear library of sequences preferred by the 2F5; the bars show the dependence of the accumulation of the sequence on its length (A) and on the K(lys)/R(arg) substitution (B).

2

3

Fig. 3. Immunogenicity of mimotopes. (A) Reactivity with the synthetic peptide NEQELLELDKWASLW of sera from mice immunized with 12-mer linear mimotopes. The mice were immunized with: (1) 3rd-round eluate; (2, 3, 4, 5) mimotopes ALSEDKWASTAS, ASSLDKWALYTS, SLDKWVLAPLAY, QTMDRWASLRWS, respectively; (6) pooled pre-immune sera. All the mimotopes were immunogenic but the clone 2 ALSEDKWASTAS was the best one (***extremely significant, oneway ANOVA, p < 0.05). (B) Reactivity with NEQELLELDKWASLW of sera from mice immunized with 7-mer cyclic mimotopes. The mice were immunized with: (1) 3rdround eluate, (2) pooled CLVLDKWAC, CMALDKWAC, CKTLDKWAC and (3) pooled CDKWAGLLC, CDKWAPPTC, CDKWALAYC (3); (#) pooled pre-immune sera representative of each group. All the mimotopes were immunogenic in a significant way (***one-way ANOVA, p < 0.05). Results of three ELISA settings each in duplicates are shown.

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Table 2 Amino acid variation in the epitope core in viruses [4,16], in library selected (Table 1) and crystallized [16] peptides. Cladesa Core epitope Library/tolerated 7mer constrained 12mer linear

Crystal

A, B, C, D, F, G

A, B, C, D, F, G

663

L + L7 N1 L18 P9 Y5 M3 S/T3 F2 H E I S Not determined

664

−

D + D27

−

K + K27

666

−

K36 R8 P1

A N Q

R H Orn Nrg Paf

A, B, C, D, F, Gc

A, B, C, D, F, G

665

D46

Ed

A, B, D, F, Gb

W + W27

−

W46

A E

Y F

A667 + A27

−

S12 V4 N2 Q

A H I L Nle

D N

Q K

(+) Tolerated; (−) not tolerated by 2F5. a Clade/neutralization (%): A(92), B(79), C(0), D(45), F(100), G(0) (from [16]). b Clade C contains at this position S, K. c Clade C contains at this position K, N, Q. d Ten-fold higher concentration of the amino acid to form the complex Orn (ornitine), Nrg (nitroarginine), Paf (p-aminophenylalanine), Nle (norleucine).

lower antibody reactivity to the peptide (Fig. 3B) than the linear mimotope peptide format (Fig. 3A). Mouse and rabbit sera were tested for their neutralization potentials by their ability to decrease HIV-1 envelope glycoprotein-CD4+ cell–cell fusion. In the preliminary step, we verified the sensitivity of this system to the 2F5 mAb that we worked with. We used ELISA to test reactivity of the mAb against a series of 15-mer overlapping peptides covering the gp41 MPER region encompassing the ELDKWAS sequence. We found the NEQELLELDKWASLW peptide was the best antigen (Fig. 4A) and used it along with the mAb 2F5 and an anti-CD4 mAb to validate the cell fusion system. Fig. 4B shows that both antibodies decreased fusion by approximately 70% (anti-CD4) and 50% (2F5) compared to the basal level (around 20% taken as 100% in the Fig. 4B). Preliminary incubation with the NEQELLELDKWASLW peptide abolished the inhibitory effect of 2F5, proving that the inhibition was due to a specific interaction of the 2F5 mAb with its epitope on the gp41 expressed in Jurkat cells (Fig. 4B). In this system, mouse sera raised against the 7mer constrained or 12-mer linear phage-mimotopes did not inhibit fusion even at a minimal 1:10 dilution (Fig. 4C). These results led us to conclude that the mouse sera did not contain neutralizing antibodies in amounts detectible by this test. Unexpectedly, the rabbit sera to the linear but not to the constrained mimotope displayed statistically significant inhibition (Fig. 4D). Verifying the possibility that the use of IgG purified from the serum will introduce higher amounts of reactive antibody to the fusion assay, we found that 10 ␮g of IgG (maximal amount that could be introduced) showed a lower reactive antibody titer than a 1:10 serum dilution (Fig. 4E). 4. Discussion 4.1. The 2F5 paratope flexibility Successful discovery of peptide mimics of epitopes by screening combinatorial libraries depends on the following library parameters: complexity, lack of bias, peptide length and whether the peptide is linear or constrained. Hence, libraries with different parameters have been screened to expand epitope mimicking options [22]. A characteristic property of the mAb 2F5 is an unusu-

ally high cross-reactivity, displayed by the ability to bind a wide array of deviations from the cognate epitope sequence ELDKWAS. The DKW core motif (some containing conservative K to R substitution) is found in all retrieved peptides. In the first comprehensive crystallographic study [16], toleration by 2F5 Fab’ of mutations in the ELDKWAS peptides in the crystal complexes were also shown. This study permits a comparison of flexibility of the interaction of 2F5 with peptides containing replacements under selection and in crystals (Table 2). It appears that the crystallization increased tolerance of the antibody paratope against epitope amino acid replacements. Thus, while all previous biochemical data demonstrated that W666 was not replaced, this position was also occupied by other aromatics in crystal structures. This left only the D664 amino acid as the absolutely conserved residue in ELDKWAS peptides. The Lys665 in crystals was replaced by a set of amino acids (including non-natural ones), whereas only arginine substituted the lysine in the biochemical experiments (noted above). Histidine at this position was never observed in peptides bound to 2F5 in solution, but was the second most frequent (after arginine) amino acid in crystal complexes. The leucine and alanine flanking the DKW (663 LeuDKWAla667 ) have distinct roles in the cognate epitope; the 663 Leu is 100% conserved and is crucial for broad neutralization by 2F5, whereas the Ala667 is substituted in some viruses without any effect on neutralization [4,16]. The different behavior of these amino acids on either side of the DKW core was of special interest to our study because they were critical for antibody recognition in the constrained peptides, but not linear peptides. In the linear peptides, these were substituted by other amino acids; however, Leu663 was more replaceable than Ala667 , which differs from the cognate epitope and the selected constrained peptides. We found the following nine different amino acids at the Leu663 position (n = 25): P9 > Y5 > M3 > S/T3 > H2 > L = I = F. Notably, the most frequently observed amino acids, proline and tyrosine, have no physiochemical similarity to leucine, and are not found in viruses because they are incompatible with survival. This highlights the extreme flexibility of the respective paratope site in interacting with this amino acid. The flexibility of this paratope site was expected to be even greater in the crystal complexes [16], but the authors bypassed this position due to its “conservation in viruses”. Should this position be investigated, the best pep-

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Y. Palacios-Rodríguez et al. / Immunology Letters 136 (2011) 80–89

A.

4.000

C. 140

7-mer

12-mer

120

Cell fusion (%)

O.D. 405 nm

3.000

2.000

1.000

100 80 60 40 20 0

1

2

Pre

1

2

3

WY

SL

DK

WN

WF

DI

TN

WL

WF DI T WN

SL WA

Pre

I

NW

FD NW SL W

WA DK EL

LL

QQ

NE

EK

QE

NE

LL

E

QE

LD

LL

KW

EL

A

DK

W

SL W

0.000

B.

12-mer

D. 120

100

7-mer

Cell fusion (%)

Cell fusion (%)

100

** 50

**

* 80 60

**

40

IgG

Pre IgG

10

10000

Pre 10

100

Pre

1000

Serum dilution

Serum dilution

E.

(65 μg/mL)

Rabbit IgG (μ g)

5 07 0.

50 0. 1

12 0. 3

5 62

0 0.

.0 10

01 0.

00

00

00 0. 00

01 00 0.

00

0.

0.

Rabbit anti-serum dilution

25

0.0

0

0.0

1.

0.2

2. 50

0.2

00

0.4

5.

0.4

00

0.6

1

0.6

1

0.8

01

0.8

0

1.0

1.0

O.D. 405 nm

10

0

mAb 2F5 + Peptide

mAb 2F5

anti-CD4

0

Basal

20

Y. Palacios-Rodríguez et al. / Immunology Letters 136 (2011) 80–89

tide candidates would be proline (PDKWA) and tyrosine (YDKWA) instead of Leu663 to determine whether the proline and/or tyrosine could allow the complex to form, as was the case with our selected peptides. According to previous studies [12] of crystal structures of complexes between the 2F5 Fab’ and peptides not carrying substitutions, Ala667 is implicated in the stabilization of 664 DKW666 (-turn structure via a hydrogen bond connection with the aspartic acid [13], but whether this is indispensable for the stability of the epitope core was not elucidated. Because Ala667 is replaced in some of the viruses and does not affect the broad neutralization ability of 2F5, it can be expected that many amino acids could be at this position in the selected peptides. However, selection experiments showed that the respective paratope sites are less flexible than Leu663 (S12 > V4 > N2 > Q = Y = G). Here, serine and valine were found in 16 out of 21 peptides and were well tolerated in crystal complexes. However, peptides containing asparagines (ANDPDKWNLTPL, MDLDKWNMKSLT) or aspartic acid (SHPGPDKWQTLP) at this position were selected, while in crystal complexes, their bulky side chains created binding difficulties that led to the prediction that amino acids with bulky side chains at this position impede crystallization [16]. This is unusual because the tolerance of the paratope sites to other epitope substitutions was higher in crystals than during selection from solution. The above comparison shows that the 2F5 Fab’ paratope in crystallization is in general more flexible than in library selection. This phenomenon awaits further explanation. The most flexible component of the 2F5 paratope is the extended CDR-H3 loop that is thought to interact with the ELDKWAS region via its base, whereas the more dynamic tip interacts with the viral membrane [27]. The role of paratope flexibility in the recognition of the natural epitope lacking substitutions was the main focus of our study. The principal finding is that this flexibility is restricted and even inhibited when the epitope is presented to the paratope in the context of a 7-mer constrained peptide at either the amino-terminal (N-CDKWAxxxC-C) or carboxy-terminal (N-CxxLDKWAC-C) ends (illustrated in Fig. 1C). Indeed, despite ample presence in the 7-mer constrained library of epitope amino acid substitution versions and peptides with a DKW and DRW core (Table 1), these peptides were discarded by the antibody. If the hypothesis that the 2F5 paratope has two binding compartments [22] is correct, the paratope predominantly or exclusively uses the multi-specific binding compartment, which requires only the DKW (or DRW) be present, during linear library screening. In contrast, during selection from the 7-mer constrained library, the epitope-specific compartment was functional, and the “multi-specific” compartment was inactive. To our knowledge, this is the first demonstration of the ability of 2F5 to reject epitope amino acid substitutions and require a longer epitope sequence. In the absence of an explanation of this paratope behavior, we hypothesize that the constrained peptides containing either DKWA on its N-terminus or LDKWA on the C-terminus adopt spatial/sequence structures resembling that of the epitope, and this structure triggers the paratope to undergo an induced-fit conformational transition to form a epitopetemplated rigidity [39] with unique binding capacity that is unable to recognize epitope-dissimilar structures. In the absence of the

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cognate epitope the paratope antigen combining site represents an amorphous and flexible precursor of this conformation whose recognition capability is unrestricted and facilitates its binding to a variety of antigens [22] The epitope (or our 7-mer cyclic peptide) triggers the final “maturation” step to a high-fidelity interaction. Constraint [19,21,22] or enhancement of alpha helices [20], has been shown to improve the affinity of peptides to the antibody. However, this is observed with many different length peptides where the epitope core sequence may be located in different regions, and this does not necessarily improve the specificity of the interaction by inhibiting tolerance to substitutions. In general, the affinity and specificity are distinct but overlapping parameters [39,40]. The most significant selections from the 7-mer constrained library demonstrate that all peptides with substitutions, DKWA and LDKWA in non-terminal regions and peptides containing DKW only (Table 1), were selected against as mutations in the viral ELDKWAS affecting the conformation critical for the membrane fusion process are selected against in nature. The following requirements were critical for the interaction with the paratope: (1) a minimum of four epitope residues (DKWA) at the N-terminus or five (LDKWA) at the C-terminus and (2) no substitutions in these sequences. However, the determinant of uppermost importance was the size of the peptide (7-mer) forming loop. Constrained libraries expressing shorter (4-mer, 6-mer) and longer (8-mer, 10-mer, 12-mer) peptides were screened with mAb 2F5 by Menendez et al. [22], but this did not result in such a uniform selection of epitope structural mimics. The antibody elicited by the gp41 immunodominant fiveresidue CSGKLIC loop-epitope selected 21 different five-residue cyclic CxxKxxC epitope mimics in the linear 12-mer library [41,42] as another example of strong antibody requirement for loop size. Unlike the CSGKLIC loop-epitope, the cognate 2F5 epitope in the gp41 is not cyclic but may be in a helical conformation [20]. It is possible that cyclic peptide structure stabilizes the amino acids in a fashion resembling their helical positioning, and it is possible that the formation of DKW ␤-turns indispensable for recognition [12–16] can only be at either the amino- or on the carboxy-terminal region. However, non-selection of peptides containing only DKW clearly points to the importance of the alanine (DKWAxxx) and both the alanine and leucine (xxLDKWA) in the formation of the structure specifically recognized by the mAb. As previously mentioned, the structural role of the alanine in stabilization of the type 1 ␤-turn was observed in the crystal 2F5 Fab’-peptide complex [13]. Why leucine is required in the selection of the xxLDKWA motif remains to be elucidated. Further investigation with structural methods of these two epitope presentations by the 7-mer cyclic peptide is needed to assess these requirements and to test the hypothesis on the formation of ␤-turns in these regions and the structural implication of the Ala and Leu epitope residues. In particular, the crystallographic study by Bryson et al. [16] demonstrated that the cyclic peptide ECDKWCS (with DKW between cysteines) could be co-crystallized with 2F5 Fab, and displayed a ␤-turn in the absence of the alanine and the leucine. The peptides selected here (CDKWAxxxC, CxxLDKWAC) and their non-selected variants with substitutions (shown in bold) (CDRWAxxxC, CDKFAxxxC, CLDRWAxxxC, CxxLDKFAC) would be of interest for similar co-

Fig. 4. Testing the neutralization potential of anti-sera in the cell-cell fusion mediated by gp120/gp41-CD4 (see Section 2). (A) ELISA showing the mAb 2F5 (0.1 ␮g/ml) reactivity with three overlapping synthetic peptides (5 ␮g/ml) showing the NEQELLELDKWASLW as the most reactive of them in the antigenicity assays (Fig. 3) and in fusion (below) assays. (B) Specificity and sensitivity of the cell-cell fusion system determined by flow cytometry: left bar shows basal level (around 20% taken as 100%) resulted from co-culture of E6 (CD4+) cells with Env+ (gp120/gp41) followed by the identification of population of syncytia by double fluorescence. Decrease of syncitya done by the anti-CD4 (22.2 ␮g/ml) and 2F5 (63.9 ␮g/ml) mAbs and abrogation of the effect of mAb 2F5 in presence of synthetic peptide NEQELLELDKWASLW is shown (**Student’s t test, p < 0.05). (C) Lack of inhibitory effect in the presence of mouse antiserum to (left panel, 12mer): whole anti-3rd round eluate (1), the ALSEDKWASTAS clone (2), and to (right panel, 7mers) whole anti-3rd round eluate (1), pooled CLVLDKWAC, CMALDKWAC, CKTLDKWAC (2) and pooled CDKWAGLLC, CDKWAPPTC, CDKWALAYC (3). Pooled pre-immune serum prepared immediately prior to the immunization(Pre). (D) Left panel (12mer): the ability of rabbit antiserum to the ALSEDKWASTAS peptide-on-phage to inhibit the fusion at 1:10 and 1:100 dilutions (**very significant, *significant, Student’s t test, p < 0.05); right panel: lack of inhibition by anti-serum to the whole 3rd round eluate (containing constrained DKWA + LDKWA peptides at 1:10 and by the purified IgG. (E) Rabbit anti ALSEDKWASTAS mimotope serum (left) and IgG (right) against NEQELLELDKWASLW peptide.

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crystallization with Fab’. Although the antibody paratope tolerated the substitution of all but one (tryptophan: LDKWA) amino acids (the leucine was omitted) in linear peptides, both in crystal and in solution, we show here that the antibody did not tolerate these substitution in the 7-mer cyclic peptide context in solution. We anticipate that the above substitution variants will not form crystal complexes because only cognate epitope amino acids can cooperatively form the cyclic structure recognized by the specific binding epitope compartment. In conclusion, mAb 2F5 possesses both multi-specificity, tolerating substitution of all the epitope ELDKWAS residues except for the aspartic acid, and strong epitope specificity, requiring at a minimum four residues. The latter potential is only displayed when the epitope sequence is presented in a certain structural context, provided here by 7-mer cyclic peptides. In this context, DKWA at the N-terminus and LDKWA at the C-terminus are the minimal epitope cores required for recognition; any other location of the epitope sequences and mutations destroy the sequence-spatial structure forming the nucleus [43] of the complex. Earlier, the importance of the alanine in binding was shown by Ala-scanning experiments in which a longer constrained synthetic peptide (LELDKWASL) [13] was tested by ELISA for interaction with 2F5, which is an assay that is different from the high-throughput library screening for ligands [22,28,44] used here. 4.2. Immunogenic potential of the described linear and constrained epitope models In light of the remarkable and distinct antigenic properties of the described peptide epitope models, the data on their immunogenicity are of interest. In the majority of studies on 2F5, investigators aimed to design an immunogen that was able to repeat the elicitation of such a broadly neutralizing antibody. The results of those immunizations were basically negative despite the presentation of the ELDKWAS (or some longer sequence) peptide on various vectors and the inclusion of a fusion peptide sequence [45] and lipids [46] as parts of the viral structures to optimize the 2F5 antigenic function (see [37] for exhaustive critical analysis of this item). Using a cell–cell fusion system, which was proven to be highly sensitive to inhibitors in our previous experiments [31–35] against 2F5 in this study, we found that mouse antiserum to the both the linear and constrained peptides did not affect fusion while the rabbit antiserum to the linear but not to the constrained peptide decreased fusion by approximately 50% and to 20% at 1:10 and 1:100 dilutions, respectively. Inhibition by mAb 2F5 was not observed in the presence of the 15-mer ELDKWAS-containing peptide competitor of the epitope (Fig. 4B). The negative result with the both serum and IgG to constrained peptide (Fig. 4C and D) is most likely due to the cyclic conformation, which, in previous experiments, also did not induce neutralizing antibodies [19,37]. A more comprehensive investigation involving affinity purification of the rabbit antibody against the linear phage-displayed peptide(s) selectable by mAb 2F5 is required to explore its anti-HIV-1 potential and verify the existence of a long CDR-H3 loop in its antigen binding region. Acknowledgments We acknowledge Carlos Larralde for guiding us through the highly professional employment of the cell–cell fusion system. The authors gratefully recognize the NIH AIDS Research and Reference Reagent Program for providing mAb 2F5, anti-CD4 mAb (clone RPAT4; BD Pharmingen), synthetic peptides, Jurkat HXBc2 and E6 cells. We thank Nayali López-Balderas and Evelyn Rivera-Toledo for technical assistance in cell-cell fusion assays. This work was supported by the Mexican National University D.G.A.P.A grant (to KG). The support by CONACYT of the Doctoral work of Y.P.-R. is appreciated.

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