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Isolation of an HIV-1 neutralizing peptide mimicking the CXCR4 and CCR5 surface from the heavy-chain complementary determining region 3 repertoire of a viremic controller

Chevigne, Andy; Delhalle, Sylvie; Counson, Manuel; Beaupain, Nadia; Rybicki, Arkadiusz; Verschueren, Charlène; Staub, Thérèse; Schmit, Jean-Claude; Seguin-Devaux, Carole; Deroo, Sabrina

Author Information
doi: 10.1097/QAD.0000000000000925

Abstract

HIV-1 entry into host cells is triggered by the recognition of cell receptors CD4 and C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) by the envelope (Env) viral glycoprotein. Env comprises two noncovalently linked subunits: gp120, which binds CD4 and coreceptors CCR5 or CXCR4, and gp41, mediating fusion [1–3]. Clinical use of entry inhibitors has emphasized the possibility to inhibit earlier stages of infection and limit the establishment of latently infected cell reservoirs [4,5].

Among the HIV-1-infected population, a minority of patients control viral replication without antiviral treatment. Based on immunological and virological criteria, these long-term survivors (LTS) can be subdivided into long-term nonprogressors (CD4+ T-cell count >500/μl), elite controllers (undetectable viral load) or viremic controllers (viral load <2000 copies/ml) [6–9]. Although persistent viremic control most probably results from a combination of not yet fully elucidated mechanisms, several studies reported the importance of host factors, such as the presence of protective human leukocyte antigen (HLA) B57 and B27 major histocompatibility complex (MHC) class I alleles or polymorphisms in CCR5 and CCR2 and a dominant strong virus-specific cytotoxic T-lymphocyte response [10–12]. The humoral immune response was considered to play a minor role and was studied mainly for preventive vaccine purposes.

The recent characterization of new neutralizing antibodies (NAbs) provided additional insight on their target recognition modes [13]. Broadly NAbs were shown to target Env by recognizing conformational or linear epitopes. Among these, the IgG1b12 broadly NAb targeting the CD4 binding site of gp120 was isolated from phage libraries of Fab fragments derived from an HIV-1 infected patient [14] and a peptide corresponding to the HCDR3 of this NAb retains the parental neutralizing activity [15] in accordance with the key role of the complementarity -determining region 3 (CDR3) in determining the antigen specificity of antibodies [16,17].

We previously described the use of phage-displayed HCDR3 fragments libraries exploiting the diversity of the whole HCDR3 repertoire to isolate nanomolar binders and bioactive peptides [18,19]. Such libraries combine the advantages of random peptides libraries and larger antibody fragments libraries: small size of the displayed fragments, intrinsic diversity conferred by their biological origin, and possibility to use immunized repertoires [19].

In the present study, we screened plasma of four LTS from the ‘Centre Hospitalier de Luxembourg’ for their ability to bind gp120 (LAI X4) in ELISA and to inhibit HIV-1 infection. One LTS (LTS-2) demonstrated strong IgG responses against gp120 and the plasma sample inhibited 80% of U87–CXCR4 cells infection at a dilution of 1/20. LTS-2 is a viremic controller [6–8] infected in 1992 and harboring an R5 provirus at the time of sampling (2006). Plasma neutralizing properties were confirmed in five longitudinal samples from 2006 to 2013 using a single cycle infection assay based on luciferase-tagged recombinant viral particles (RVA) [20,21]. LTS-2 genotyping revealed the absence of the CCR5 delta 32 deletion, the CCR2 V64I and the SDF1 G801A polymorphisms and combination of HLA A*02/*11, B*27/*35, and C*02/*04 alleles. Although B*35 is known to be associated with rapid disease progression, B*27, A*02, and A*11 are in contrast linked to a delayed progression of HIV-1 infection [22–25]. In the absence of cryopreserved blood cell samples, further investigation on protective cytotoxic T-lymphocyte responses against HIV-1 was not possible. As previously described [18,19], the IgM and IgG HCDR3 repertoires of LTS-2 were PCR amplified with primers located in the framework regions 3 and 4 of the VH gene, cloned in a phagemid vector, and fragments corresponding to residues 86 to 109 and including the HCDR3 sequence (positions 95–102) (Kabat numbering) were expressed on phage surface. Fragment length in the HCDR3 repertoire was analyzed using Genescan method and revealed a Gaussian-like pattern of sizes ranging from 3 to 18 residues for both LTS-2 IgM and IgG repertoires, as we previously observed in healthy donors [19]. These data indicate that the LTS-2 HCDR3 repertoire is not biased toward a certain length and corresponds to a HIV-1 negative repertoire. These repertoires were screened on recombinant X4 virus gp120 protein (LAI) immobilized on magnetic beads. Biopanning with IgM library allowed the isolation of 15 different sequences out of 60 sequenced positive clones. Among these 15 sequences, 11 were unique and four sequences occurred at frequencies of 36, 6, 4, and 3. From the IgG biopanning, the 57 positive clones corresponded to a unique sequence (LRTV-1), which was identical to the most frequently isolated IgM HCDR3 sequence (36/60). The specificity of the LRTV-1 sequence was confirmed by phage ELISA and gp120 binding was enhanced by CD4 (Fig. 1a). A cyclic peptide corresponding to LRTV-1 HCDR3 (89-CAK-VPQVDFWRRIFDY-WCRGTTV-106) was synthesized. LRTV-1 protected MT-4 and MT4-CCR5 cells from infection by X4 (IIIB) and R5 (BaL) viruses with IC50 = 45 ± 1.6 μmol/l (132 μg/ml) and 58 ± 1.8 μmol/l (171 μg/ml), respectively. The antiviral activity was further confirmed using RVA with HIV-1 X4 (NL4.3, IC50 values of 21 ± 2 μmol/l) (62 μg/ml), or R5 (NLAD8, 54 ± 4 μmol/l) (159 μg/ml) Env, whereas LRTV-1 had no effect on vesicular stomatitis virus glycoprotein pseudovirus activity (Fig. 1b) [20]. No inhibition of viral infection was observed with two different scrambled LRTV-1 sequences. Further analysis using a panel of 11 recombinant viruses harboring envelope of primary X4 and R5 isolates of different tiers (1, 2, and 3) and subtypes (B, C, or AG) showed a protective activity of peptide LRTV-1 ranging from 21 to 143 μmol/l (Table 1 Supplementary Data, http://links.lww.com/QAD/A810). LRTV-1 peptide was respectively 21, 124 620, and 23 800-fold less potent than 2F5 (2.9 μg/ml) [26], 2G12 (0.5 μg/ml) [27], B12 (0.1 μg/ml) [14], and T20 (0.0026 μg/ml) in inhibiting the infection of U87–CXCR4 cells by HIV-1 X4 (NL4.3) virus [28]. LRTV-1 showed sequence homology (7/13 identical residues, positions 95, 96, 99, 100, 100A, 101, 102, and 103) with an HCDR3 sequence (Abysis JN577803) previously identified during the large-scale sequencing of IgG mRNA transcripts of a cohort of healthy donors and HIV-1 infected patients, however, no information about the neutralizing properties of the corresponding antibody was reported [29]. Sequence alignment of LRTV-1 with coreceptors CXCR4 and CCR5 revealed a high degree of similarity with the C-terminus of the second extracellular loop of CXCR4 (ECL2-X4) (11/23 residues, including five identical residues) and with the N-terminus of CCR5 (7/23) (Fig. 2a) suggesting that LRTV-1 impairs the interaction between gp120 and coreceptors, in agreement with CD4 dependency of LRTV-1 binding on gp120. ECL2 of CXCR4 and the N-terminus of CCR5 are indeed major gp120 binding determinants at the coreceptor surface and we have recently shown that peptides derived from ECL2-X4 (176–202) have antiviral properties against X4-strain NL4.3 (IC50 = 9.3 ± 0.3 μmol/l) (Fig. 1b) [30–37]. These results are consistent with reporting of CCR5 surface mimicry by gp120 specific antibodies CDRs [38,39]. To precisely identify the LRTV-1 residues interacting with gp120, an alanine scan of the 23 residues was undertaken. Mutations of the two cysteine residues (C92A and C104A) involved in the HCDR3 disulfide bridge as well as of their adjacent residues (F91A, A93F, and W103A) had a major impact on binding (Fig. 2b), indicating that cyclization of LRTV-1 is most likely crucial for its interaction with gp120. Mutations of residues P96, F100, W100A, I100D, F100E, and D101 (Kabat numbering) within the HCDR3 sequence decreased binding of LRTV-1 to gp120. Most of these residues are conserved or share biophysical properties with residues in ECL2-X4 (I185, C186, L194, F195, and V198-Q200) and N-terminus of CCR5 (Y3, V5, P8, I12, and E18). As an additional proof of coreceptor surface mimicry, a 25-fold molar excess of peptide LRTV-1 reduced binding of peptide ECL2-X4 to the gp120-CD4 complex by more than 30% (Fig. 1a inset). Although it is difficult to evaluate whether LRTV-1 HCDR3 sequence is part of the paratope of LTS-2 NAbs, we showed using ELISA that the LRTV-1 peptide, but not the corresponding scrambled peptides, reduces the binding of LTS-2 IgGs to gp120-CD4 complex by 30% (Fig. 1 Supplementary Data, http://links.lww.com/QAD/A809). This observation suggests that part of the IgGs present in LTS-2 plasma recognize the same gp120 epitope as LRTV-1 peptide. In conclusion, screening phage-displayed IgG and IgM-derived HCDR3 repertoires of a viremic controller presenting NAbs resulted in the identification of a HCDR3-derived peptide able to bind gp120 and to inhibit HIV-1 infection of both R5 and X4 viruses including primary isolates. Importantly, the long-lasting plasmatic neutralizing activity of LTS-2 patient indicates that the limited viral replication persisting in this patient allows a sufficient antigenic stimulation to elicit NAbs. This is in accordance with NAb responses directed against Env variants previously observed in long-term controllers [40] and the strong therapeutic potential of NAbs for HIV cure in animal models [41–43]. It also evidences that HCDR3 peptides with their small size, high affinity, and selectivity offer a promising alternative to whole antibodies, single-chain variable fragments, and single variable domain derived from heavy-chain only antibodies (Nanobodies) to target difficultly accessible protein sites and thus, represent a new source of highly diverse therapeutic peptides. However, the potency (low micromolar range) and stability of LRTV-1 peptide could be largely improved by incorporating D-amino acids or other chemical derivatives or improving delivery mode using nanoparticles [44–49]. To this end the mutational analysis conducted in this study provides valuable positional information for such further improvements. Finally, according to its inhibitory effect on both R5 and X4 viruses, LRTV-1 peptide might become useful in the search of new dual CCR5/CXCR4 HIV-1 inhibitors.

Fig. 1
Fig. 1:
CD4 binding dependency of phage displayed LRTV-1 cyclic peptide to gp120 and inhibition of HIV-1 infection by LRTV-1 and ECL2-X4 peptides.(a) Specificity of the most frequently isolated phage clone (LRTV-1) for gp120 in the presence or absence of soluble CD4 (sCD4). Binding was determined by ELISA on immobilized gp120 with or without 2 molar equivalent of sCD4, sCD4 alone, or control glycoprotein (ovalbumin). Two-fold dilutions of phage displaying LRTV-1 or irrelevant HCDR3 sequence (starting at 1.1011 phage per well) were added. Phage binding to immobilized proteins was detected using an anti-M13 IgG conjugated to horseradish peroxidase. The experiment was performed twice and resulted in equivalent profiles. One representative of two is shown. Inset: Competition between biotinylated ECL2-X4 (10 μmol/l) and nonbiotinylated LRTV-1 or irrelevant HCDR3 (250 μmol/l) peptides for binding to gp120-CD4 complex. Binding of biotinylated ECL2-X4 peptide was detected using streptavidin conjugated to horseradish peroxidase. (b) Inhibition of NL4.3 and NLAD8 recombinant virions and VSV-g pseudoparticles by LRTV-1 peptides. U87.CXCR4 or U87.CCR5 cells were infected with recombinant HIV particles harboring the CXCR4 or CCR5-using envelope protein (NL4.3 or NLAD8) and of VSV-g pseudovirions in the presence of serial three-fold dilutions of LRTV-1, scrambled (scrbl) (LRTV-1scrbl1: YRFCWFKGVIYDVDVARPQRCWF,LRTV-1scrbl2:WVRCYYDVKFGWIFAQRDPRCFV), or ECL2-X4 peptides. Viral infection was evaluated by measuring luciferase activity in cell lysates after 4 days. Data are presented as percentage of infection obtained in absence of peptide. All experiments were performed in triplicate and are presented as average ± standard deviation. Toxicity of peptides was tested in the same assay by measuring cell survival in uninfected U87.CD4.CXCR4 cultures exposed to the peptides using an MTT assay. VSV-g, vesicular stomatitis virus glycoprotein.
Fig. 2
Fig. 2:
Sequence comparison between HCDR3 LRTV-1 and ECL2 of CXCR4, and N-term of CCR5 and characterization of the gp120 interacting residues of LRTV-1.(a) LRTV-1, ECL2-X4, and N-term-R5 sequence alignment. Identical residues are colored red, whereas nonconserved residues presenting similar biochemical properties in the three sequences are colored blue. Residues similar in LRTV-1/ECL2-X4 or LRTV-1/N-term-R5 pairs are colored orange and green, respectively. The HCDR3 sequence is numbered according to Kabat numbering. (b) Alanine scanning of the interaction between LRTV-1 peptide displayed on phage (2 × 1012 phage per well) and gp120. Data are represented as an average of triplicate measurements of the absorbance recorded with an anti-M13 antibody conjugated to horseradish peroxidase ± standard deviation. The experiment was conducted twice and resulted in equivalent profiles. WT represents signal measured with phage displaying wild-type LRTV-1 HCDR3. Alanine at position 93 was replaced by a phenylalanine.

Acknowledgements

The work was supported by the ‘Fondation Recherche sur le SIDA’ and the Ministry of Research of Luxembourg, grants CRP-20070115 (GPCR47-GPCR47bis), 20101002 (Eumimo). Authors thank Dr Perez-Bercoff for advice regarding RVA and the NIH AIDS Reagent Program and its contributors (see Table 1 Supplementary Data, http://links.lww.com/QAD/A810) for panel of subtypes C and B reference Env clones.

Author contributions: A.C., C.D., and S.D. designed the experiments. Sy.D., M.C., N.B., A.R., and C.V. performed the experiments. A.C. analyzed the data. A.C., Sy.D., and C.D. wrote the paper. J.C.S., T.S., and S.D. provided patient samples, clinical data, and/or revised the publication.

Conflicts of interest

There are no conflicts of interest.

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Keywords:

CCR5; CXCR4; heavy-chain complementary determining region 3; HIV-1; neutralizing antibody; phage display; viremic controller

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