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J Virol. 2006 Jun; 80(11): 5552–5562.
PMCID: PMC1472165

Sequential Immunization with V3 Peptides from Primary Human Immunodeficiency Virus Type 1 Produces Cross-Neutralizing Antibodies against Primary Isolates with a Matching Narrow-Neutralization Sequence Motif


An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the “PGR” motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.

In humans, antibodies, whether actively induced or passively transferred, neutralize viruses and therefore protect against viral diseases like hepatitis and influenza (6, 15). However, the specific antibodies that confer protective immunity against human immunodeficiency virus type 1 (HIV-1) infection are not well known, since most primary strains of HIV-1 are relatively resistant to neutralization (40, 47). Studies with recombinant monomeric gp120 have not been successful at predicting the neutralization of primary isolates (12, 39). However, considerable progress in understanding HIV pathogenesis, namely, in determining that both antibody- and cell-mediated immune responses are likely responsible for controlling viral load, has recently been made (10, 44, 60). With regard to the role of neutralizing antibody responses in HIV-1 infection, broadly reactive neutralizing antibodies such as 2G12 (54), 2F5 (45), and 4E10 (5) have been proven to suppress immune deficiency virus infection in macaques (14, 38) and humans (62). However, it remains to be seen whether high-titered and cross-reactive neutralizing antibodies will be produced by active immunization with a novel viral antigen.

In this study, we attempted to develop a sequential immunization strategy that has proven to be effective at eliciting neutralization antibodies to primary HIV-1 by targeting the HIV-1 Env V3 neutralization epitope site as a model antigen. Previously, we demonstrated that the Gly-Pro-Gly-Arg (GPGR) core sequence at the tip of the V3 region of gp120 is relatively conserved in both field and clinical isolates of HIV-1 clade B, while the flanking regions of the V3 tip are more variable (1, 66). While in theory, high-affinity antibodies that recognize the relatively conserved GPGR epitope could potentially neutralize many strains of HIV-1 clade B, in practice, such antibodies in sera from HIV-infected individuals show little neutralization activity in vitro (2), suggesting that the immunogenicity of the GPGR sequence is similarly low. To overcome this problem, we sequentially immunized mice with V3 peptides from HIV-1 clade B field isolates, resulting in the induction of cross-reactive antisera that strongly bound to V3 peptides from homologous and heterologous primary isolates. Furthermore, a cross-reactive neutralizing monoclonal antibody (MAb), C25, was established. KD-247, a reshaped MAb derived from a C25 gene that had been reshaped to a humanized antibody, also efficiently neutralized primary isolates of HIV-1.

Although anti-V3 antibodies elicited by active immunization with the V3 peptide and HIV Env have been reported to neutralize HIV-1 both in cell culture and in animal challenge studies, these antibodies have not yet been fully exploited because they are type specific (13, 29, 33, 46). In contrast, anti-V3 MAbs generated by heterohybridoma (22) using peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals have recently been shown to contain cross-neutralizing anti-V3 MAbs and neutralize primary isolates (11, 18, 19, 21, 23, 25). Furthermore, it has been suggested that the neutralization sensitivity of primary isolates is regulated by the V1/V2 domain of Env gp120 (48). In this study, we demonstrate that cross-neutralizing anti-V3 antibodies against primary isolates can be produced via sequential immunization with the V3-neutralizing epitope peptides of HIV-1. Furthermore, we discuss how sequential immunization with Env peptides including a neutralization epitope could pave the way for the generation of cross-reactive neutralization antibodies.



C3H/HeN mice were bred at The Chemo-Sero-Therapeutic Research Institute Experimental Animal Center and used when they were between 4 and 8 weeks of age for immunization and generation of MAbs. The study was conducted with the approval of an institutional committee for biosafety and animal welfare. The mice were housed in accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science under the Japanese Law Concerning the Protection and Management of Animals (59) and were maintained in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of Kaketsuken, Japan.

Isolation of MAb C25 and sequencing of the V region gene.

V3 sequences from HIV-1 clade B isolates were determined as previously described (50) and used to generate peptides SP1, SP17, SP11, SP14, SP12, and SP13 for sequential immunization of mice (Fig. (Fig.1A).1A). Of the six V3 peptides generated, only the SP1 peptide was likewise repeatedly administered to another group of mice as a reference for the sequential immunization. A cysteine residue was added to each peptide for coupling to the keyhole limpet hemocyanin, and the peptides were then emulsified with complete and incomplete Freund's adjuvant at a concentration of 100 μg/ml. Peptides were administered at 1-week intervals by intraperitoneal injection, with the exception of SP13, which was injected intravenously 1 week after the SP12 inoculation. Hybridomas were generated from mice with antisera that are highly cross-reactive to HIV-1, as described previously (32), and clone C25 was selected by a combination of enzyme-linked immunosorbent assays (ELISAs) and neutralization assays (26). RNA was extracted from the C25 hybridoma by conventional methods and used as a template for the synthesis of first-strand cDNA. Primers used for PCR amplification of the 5′ mouse immunoglobulin (Ig) V regions and the 3′ J region were designed based on the nucleotide sequence database for mouse Ig as classified previously by Kabat et al. (30). The PCR primers used to obtain the mouse heavy-chain Ig gene were MHL4ver.4 (5′-TTCGAAGCTTGCCGCCACCATGGAATGGAGCTGGGTCTTT-3′) and MHCver.1 (5′-GGATCCCGGGAGTGGATAGACCGATG-3′), and the primers used to obtain the mouse light-chain Ig gene were MκL7ver.2 (5′-ACTAGTCGACATGGGCATCAAGATGGAGT CACAGATTCTGG-3′) and MκCver.1 (5′-GGATCCCGGGTGGATGGTGGGAAGATG-3′). Restriction enzyme sites for HindIII (V region) and BamHI (J region) were incorporated into the primers to facilitate cloning. PCR amplification was performed using cDNA as a template and the V and J region primers, as described above. The resulting PCR products were then cloned into the HincII site of pUC18, and the sequence of the inserted V region gene was confirmed by the Sanger dideoxy-mediated chain termination method.

FIG. 1.
Generation of cross-reactive antibodies by sequential immunization with different V3 peptides originating from HIV-1 clade B field isolates. (A) Aligned V3 sequences used for sequential immunization and ELISA screening. (B) Binding antibody titers of ...

Preparation of humanized MAb KD-247.

Transfer of the complementary determining regions (CDRs) and partial framework regions (FRs) of C25 into the human V regions was carried out according to established methods for preparing a humanized MAb. In brief, CDRs and a part of the FRs of the VH region of C25 were transferred into the VH FR of human subgroup I, while CDRs of the VL region of C25 were transferred into the FR of the human κ-chain REI. CDR grafting was performed by PCR-based in vitro mutagenesis. Computer-aided molecular modeling was used to design the initial CDR-grafted and resurfaced versions of the constructs, which were then cloned into a glutamine synthetase expression vector (GS system; Lonza Biologics PLC, Berkshire, United Kingdom). The vector was transfected into NS0 myeloma cells (Lonza), and glutamine-independent transfectants were isolated. Cells producing KD-247 were expanded in large-scale cultures, and the antibody was purified from the culture supernatants by ion-exchange and affinity chromatography.

Surface plasmon resonance.

The kinetic constants for the binding of MAbs to the SP1 peptide were determined by surface plasmon resonance measurements (31) using a BIAcore 1000 system (BIAcore AB, Uppsala, Sweden). The SP1 peptide was coupled with biotin-PE-maleimide (Dojindo Laboratories, Kumamoto, Japan) through a thiol-containing cysteine residue that was deliberately placed at the carboxyl terminus. Biotinylated SP1 was then coupled to streptavidin on the surface of a sensor chip (Sensor Chip SA; BIAcore AB, Uppsala, Sweden) by injection of 10 μl of a 100-ng/ml solution in HBS-EP buffer (BIAcore). Approximately 5 resonance units of SP1, a value recommended by the manufacturer, were immobilized on the chip. MAbs KD-247, Rμ5.5, and Cβ1 (41) were diluted in HBS-EP buffer to concentrations of 40, 60, 80, 100, and 160 nM, and 20 μl of each MAb was injected over the immobilized antigen at a flow rate of 20 μl/min. At the end of the association phase, the dissociation of each MAb was measured over an 8-min period at the same flow rate. The surface of the chip was regenerated with 20 μl of 10 mM HCl. Kinetic rate constants were calculated from data generated at each concentration of MAb using BIAevaluation version 3.0 software (BIAcore). The model for simultaneous ka/kd determination was selected for bivalent analysis because the samples were regarded as intact antibodies.

Pepscan analysis.

The following peptides were synthesized by an Epitope Scanning kit (Chiron Mimotopes Pty., Ltd., Victoria, Australia) according to the Pepscan method (17) and examined for binding activity to KD-247: IHIGPGRAFY, IRVGPGRTLY, IRVGPGRAIY, and LSVGPGRSFY (corresponding to the V3 tip of HIV-1 strains MN, NI53, NI54, and TM2, respectively). We also determined which peptides had consecutive amino acids deleted, which had an overlapping series of peptides of 4 to 10 amino acids in length derived from the HIV-1MN V3 tip sequence IHIGPGRAFY and which had each of their amino acids consecutively replaced by one of the 19 naturally occurring amino acids. The reactivity of KD-247 with bound peptides was determined by a peptide-based ELISA. Polyethylene rods with bound synthetic peptides were precoated with 2% bovine serum albumin-0.1% Tween 20 in phosphate-buffered saline for 1 h at room temperature, followed by a reaction with 2 μg/ml of KD-247 in the precoated buffer overnight at 4°C. After the reaction of KD-247 with a peroxidase-labeled goat anti-human kappa antibody (Southern Biotechnology Associates, Birmingham, AL), the presence of the MAb was detected by reaction with peroxidase substrate, and the resultant color was read at an optical density at 450 nm.

In vitro virus neutralization assay.

The international reference antibodies 1006-15D, 694/98D, 1331-D, and 1367-D were kindly provided by Susan Zolla-Pazner, New York University School of Medicine, New York, NY, and 447-52D was obtained from the AIDS Research and Reference Reagent Program, National Institutes of Health, Rockville, MD (20, 21, 42). Rμ5.5 and Cβ1 (41) were also used as reference antibodies. An MT-4 cell-based virus neutralization assay was performed to screen for hybridomas and to evaluate MAbs as described here. Briefly, serial dilutions of test samples were plated onto eight wells of a microtiter plate. HIV-1 (100 50% tissue culture infective doses [TCID50s]) was added to each well and incubated at 37°C in a CO2 incubator for 1 h. One hundred microliters of MT-4 cell suspension (1 × 105 cells/ml) was then added to each well and cultured at 37°C in a CO2 incubator for 5 days without agitation. After cultivation of MT-4 cells for 5 days, the cells were dissociated in each well, and the presence or absence of syncytium was examined under a light microscope. Neutralizing activity (90% inhibitory concentration [IC90]) was defined as the minimum effective concentration of antibody that inhibited infection in all wells at the same concentration. The neutralization assay using the MT-4 cell line was conducted for the screening of hybridomas and the evaluation of MAbs against laboratory and primary isolates. Laboratory-adapted HIV-1MN (H9/HTLV-III MN) and HIV-1IIIB (H9/HTLV-IIIB NIH1983) were obtained from the AIDS Research and Reference Reagent Program. Virus stocks of primary HIV-1 clinical isolates were prepared by coculturing phytohemagglutinin (PHA)-activated human PBMCs from both HIV-seropositive and healthy individuals, as described previously (11, 28).

GHOST cell neutralization assays were performed to study the neutralization activity of antibodies against HIV-1 as previously described (7, 58). Briefly, both GHOST-X4 and -R5 cells, which express the CXCR4 and CCR5 coreceptors, respectively, were used as target cells for viral infection. Cells used in neutralization assays were analyzed by FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA), and 15,000 events were scored. The cutoff value was considered to be the mean number of fluorescent GHOST cells used for background control plus 2 standard deviations. A primary clinical isolate, HIV-1MNp, was kindly provided by J. Sullivan of the University of Massachusetts Medical School, Worcester, MA. The virus was confirmed as one of the neutralization-resistant isolates by Susan Zolla-Pazner (7). Laboratory-adapted HIV-189.6 and HIV-1MN were obtained from the AIDS Research and Reference Reagent Program. Simian/human immunodeficiency virus (SHIV) strain C2/1 was provided by K. Shinohara of the National Institute of Infectious Diseases (NIID), Tokyo, Japan (52, 56). SHIV strain 86.9 PD was kindly provided by Y. Lu of the Virus Research Institute, Cambridge, MA.

PBMC-based virus neutralization assay.

Primary isolates were cultured with PHA-activated PBMCs in the presence of 40 units/ml of human interleukin-2 (Shionogi Pharmaceutical Co., Osaka, Japan) for 7 days, virus stocks were titrated on PHA-activated normal PBMCs, and the TCID50 of each virus was determined (28). The primary isolates of HIV-1JR-CSF and the CS series were provided by Y. Koyanagi, Kyoto University Virus Research Institute, Kyoto, Japan, and JCI series isolates were also used. In vitro virus neutralization assays using diluted serum antibodies were performed as previously described (9, 28). In brief, 50 μl of various concentrations of serum IgG was preincubated with 50 μl of 100 TCID50s of each virus strain in triplicate for 1 h at 37°C in a round-bottomed 96-well plate (Costar, New York, NY).One hundred microliters of 105 PHA-activated PBMCs originating from a single batch was added to that mixture for 1 h. After being washed, the cells were cultured in the presence of human interleukin-2 every day for approximately 7 to 21 days, with the period of culture depending on the property of each virus, and the amount of HIV was then measured by a p24 antigen ELISA (Dainabot, Tokyo, Japan). The neutralization titer is given as the concentration of serum IgG antibody or the reciprocal of serum dilution that reduced p24 antigen production by 90% (IC90) or 50% (IC50) compared to control wells with purified serum IgG from healthy individuals and preimmune mouse sera.


Sequential immunization with synthetic V3 peptides from HIV-1 clade B field isolates generates a cross-reactive antibody response.

Our previous study, which described anti-V3 antibody production, used single HIV-1 Env V3 peptides and produced homologous neutralizing antibodies. To find a more appropriate procedure to elicit a cross-reactive antibody, we opted for sequential immunization using six synthetic peptides (SP1, SP17, SP11, SP14, SP12, and SP13) overlapping the tip of the V3 region of gp120 from both field and laboratory HIV-1 strains (Fig. (Fig.1A).1A). Antibodies present in sera from the immunized mice exhibited high levels of binding to all the six homologous peptides and were highly cross-reactive with peptides (SP6, SP9, and SP10) derived from heterologous HIV-1 clade B field isolates containing the V3 core GPGR sequence (Fig. (Fig.1B).1B). In contrast, repeated immunization with HIV-1MN Env V3 SP1 peptide alone generated antibodies with specificity only for SP1 (Fig. (Fig.1C).1C). Thus, sequential immunization using synthetic peptides derived from the V3 tip of HIV-1 gp120 generated site-specific and cross-reactive binding antibodies.

In order to analyze the neutralizing ability of the mouse antisera, a PBMC-based virus neutralization assay was performed using T-cell-line-adapted (TCLA) HIV-1MN, CXCR4-tropic primary HIV-1MNp, and CCR5-tropic primary HIV-1JR-CSF. Both types of immunizations successfully induced high neutralization titers with IC50 values of 450 in immune sera from sequentially immunized animals and with IC50 values of 300 in immune sera from mice receiving repeated immunizations of single SP1 peptides against HIV-1MN (Fig. (Fig.2A).2A). Furthermore, the immune sera from the mice sequentially immunized with the peptides efficiently neutralized HIV-1MNp with an IC50 titer of 110 and an IC90 titer of 40 and neutralized HIV-1JR-CSF with an IC50 of 200 and IC90 of 60 (closed squares in Fig. 2B and C, respectively). In contrast, the immune sera from mice immunized with the single SP1 peptide had no ability to neutralize the HIV-1MNp and HIV-1JR-CSF isolates (closed triangles in Fig. 2B and C, respectively). Preimmune and healthy control sera had no ability to neutralize the above-described three viruses. In summary, sequential immunization with representatives of Env V3 peptides produced high levels of cross-reactive neutralization responses in the immunized animals.

FIG. 2.
Neutralization of HIV-1MN and clinical isolates HIV-1MNp and HIV-1JR-CSF by sera from sequentially immunized mice with diverse V3 tip peptides but not by sera from mice repeatedly immunized with a single SP1 peptide. Shown are data from neutralization ...

Production and neutralizing capacity of mouse MAb C25 and its humanized MAb, KD-247.

It is important to determine whether the cross-reactive neutralization responses could be due to the effect of cross-reactive neutralization antibodies and to compare the data with those of established reference neutralization antibodies (7, 21). We first used the immunized mice with highly cross-reactive antibody responses and selected a hybridoma, named C25, which was reactive with heterologous V3 peptide (Fig. (Fig.1D).1D). MAb C25 reacted efficiently with the synthetic V3 peptides from multiple heterologous strains of HIV-1 at concentrations of less than 0.01 μg/ml; however, SP17 (Fig. (Fig.1D,1D, open circles) was less reactive, and SP14 (Fig. (Fig.1D,1D, open triangles) showed no response at all. Furthermore, MAb C25 was highly reactive with all three clinically isolated heterologous V3 peptides (SP6, SP9, and SP10) (Fig. (Fig.1D).1D). In short, sequential immunization proved to be effective at eliciting cross-reactive binding antibodies against the V3 tip epitopes of HIV-1. Furthermore, since C25 possessed cross-reactive neutralization abilities as described Table Table1,1, we produced a humanized antibody using the gene of the C25 mouse hybridoma. The CDRs and the partial FRs in the C25 gene were transferred into the human V region gene (data not shown) to produce a stable reshaped antibody clone known as KD-247. To reshape KD247, we aligned the amino acid sequences deduced from the genes for C25 and KD-247 with the FR of the REI VL and the FR of the subgroup I VH regions (Fig. (Fig.33).

FIG. 3.
Design to synthesize an amino acid sequence that encoded the reshaping antibody KD-247. Amino acid sequences of VH and VL regions of human IgG, KD-247, and C25 are shown. Boxes indicate amino acids in the CDRs and a portion of the FR segments in the reshaped ...
Neutralizing activities of C25 and KD-247 against HIV-1 clade Ba

To assess the neutralizing abilities of C25 and KD-247, we evaluated their neutralization abilities against reference laboratory and clinical isolates by using MT-4 cell-based and PBMC-based neutralization assays. As shown in Table Table1,1, we selected those target viruses that possessed a GPGR sequence in the Env V3 tip region. C25 and KD-247 similarly neutralized laboratory and primary isolates of CXCR4 and CCR5, with the exception of HIV-1IIIB, which has an insertion of QR in front of the glycine residue of the V3 tip sequence. As expected based on previous research, MT-4 cell- and PBMC-based neutralization assays showed that various concentrations (0.1 to 34.0 μg/ml of antibodies for IC90) of both the C25 and KD-247 antibodies were capable of neutralizing target viruses that possessed a GPGR sequence in the V3 tip sequence. To evaluate the neutralization abilities of KD-247, we compared its neutralization activity with those of established reference antibodies by GHOST cell neutralization assays using GHOST-X4 and GHOST-R5 cells (7) expressing the chemokine receptors CXCR4 and CCR5, respectively. As shown in Fig. Fig.4,4, KD-247 efficiently neutralized HIV-1MNp on GHOST-X4 cells with an IC90 of approximately 5 μg/ml, while neither normal human IgG nor a similar reshaping anti-V3 antibody, Rμ5.5, which recognizes the V3 tip sequence IHIGPGRAFYT, neutralized HIV-1MNp. Thus, although the V3 tip sequence of HIV-1MNp (RIHIGPGRAFYTTKN) (Table (Table2)2) was identical to the V3 sequence of Rμ5.5 (underlined), Rμ5.5 did not neutralize HIV-1MNp, but KD-247 did. These results suggest that the neutralization epitope of HIV-1MNp may be narrower than that of the V3 sequence of Rμ5.5 and that KD-247 may recognize the narrow-neutralization epitope of the V3 tip sequence. Testing of the laboratory isolate HIV-1MN by the GHOST cell assay confirmed that KD-247 neutralized HIV-1MN efficiently, with an IC90 and IC50 of 1.0 and 0.1 μg/ml, respectively. HIV-1AD8, SHIV 89.6, and SHIV C2/1, whose V3 tip sequences were confirmed to be KSIHIGPGRAFYT, RLSIGPGRAFYA, and RLSIGPGRAFYA, respectively, were less sensitive to neutralization by KD-247, with IC90 values of 10, 5, and 5 μg/ml, respectively. In contrast, those three viruses were not neutralized by the other previously reported anti-V3 neutralizing MAbs, Rμ5.5 and Cβ1.

FIG. 4.
Neutralization of HIV-1MNp by KD-247 in GHOST-X4 cells. Each dilution of MAbs KD-247 and Rμ5.5 was tested in triplicate, and results are expressed as the means of three different experiments. Normal human IgG (NHIG) was used as the control IgG ...
Neutralizing activity of KD-247 against R5 and R5/X4 dual-tropic viruses by GHOST cell assaya

To further substantiate that KD-247 neutralizes primary isolates, we also tested seven primary isolates by comparing them to the following international reference antibodies: anti-V3 MAb 447-52D, anti-V3 MAb 1006-15D, anti-V3 MAb 694/98D, anti-C5 MAb 1331-160, and anti-gp41 MAb 1367-D (Table (Table3).3). KD-247 most efficiently neutralized HIV-1 clade B primary isolates from the AIDS Research and Reference Reagent Program, NIAID, and from the HIV Isolation Project, NIID, with IC50s at various concentrations, from 0.78 to 34.26 μg/ml, although it did not prove effective against the N-NIID isolate, which possessed a GPGR sequence in the Env V3 tip region. These results demonstrate that KD-247 more effectively neutralizes primary isolates with both CXCR4 and CCR5 tropism than do any of the reference antibodies (Tables (Tables22 and and33).

Fifty-percent neutralization activity on GHOST/R5 or GHOST/X4a

Properties of the anti-HIV envelope V3 MAb KD-247 that neutralize viral infection.

To more precisely address the neutralization mechanisms of KD-247, we investigated its biochemical properties.

(i) Peptide affinity.

The affinity of KD-247 for the SP1 peptide was evaluated by surface plasmon resonance-based measurements using a BIAcore instrument and then compared to the affinity of Rμ5.5 and Cβ1 for HIV-1MN and HIV-1IIIB, respectively (41). Sensorgrams showed that Cβ1 bound slightly to SP1, while KD-247 bound more than did Rμ5.5 (Fig. (Fig.5).5). The equilibrium dissociation constants (KDs) of KD-247 and Rμ5.5 were 1.3 × 10−9 M (ka, 1.3 × 105 M−1 s−1; kd, 1.7 × 10−4 s−1) and 2.9 × 10−10 M (ka, 1.0 × 105 M−1 s−1; kd, 2.9 × 10−5 s−1), respectively.

FIG. 5.
Sensorgram overlays for the binding of KD-247, Rμ5.5, and Cβ1 to the SP1 peptide.

(ii) Epitope mapping.

We tested KD-247 for epitope mapping by reacting the antibody with decamer peptides derived from the V3 tip sequences of three different clinical HIV-1 isolates (NI53, NI54, and TM2) and HIV-1MN. Deletion of amino acids lying in the central region of each peptide resulted in a reduction in KD-247 binding, while deletion of amino acids near either terminal had little effect (Fig. (Fig.6A).6A). To determine the shortest reactive peptide, KD-247 was tested for binding activity against a series of overlapping peptides that varied in length from 4 to 10 amino acids. For optimal binding, KD-247 required peptides of five or more amino acids containing an IGPGR sequence (Fig. (Fig.6B).6B). In addition, sets of peptide analogues that differed from the original decamer peptide by only one amino acid were synthesized. Each amino acid within a given peptide was replaced in turn by one of 19 naturally occurring amino acids, and the resulting peptides were tested for reactivity against KD-247 (Fig. (Fig.6C).6C). The amino acids I, H, A, F, and Y in the flanking region of the core sequence could easily be replaced by many other residues without the loss of KD-247 binding (Fig. (Fig.6C,6C, panels a, b, h, i, and j). Although a few amino acids in the core PG sequence could be replaced (Fig. (Fig.6C,6C, panels e and f), the arginine residue was found to be critical for maintaining KD-247 binding at levels equivalent to those of the original peptide (Fig. (Fig.6C,6C, panel g). In summary, KD-247, a humanized antibody with high affinity (KD of 1.3 × 10−9 M), recognized the PGR of the narrow V3 tip sequence and should therefore act as an effective neutralizing antibody.

FIG. 6.
Pepscan with deletion (A), overlapping (B), and replacement (C) peptides. KD-247 binding activity for the peptides is proportional to the value of the optical density (O.D.) at 450 nm. Hatched bars indicate the reactivity of the original sequence without ...


Sequential immunization with multiple V3 peptides of HIV-1 Env is desirable in that it can evoke cross-reactive HIV-1-neutralizing antibodies, while repeated immunizations with a single peptide elicit only a non-cross-reactive and virus-specific neutralizing antibody response. Of these two types of antibody responses, only the cross-reactive one produced by sequential immunization neutralized CCR5-tropic primary and other heterologous isolates. Because it appears to raise an effective antibody specific for the HIV-1 Env neutralization epitope, this sequential immunization regimen may show considerable promise for use in vaccine development by an active immunization procedure.

A single C4-V3 peptide immunogen from HIV-189.6 was shown to partially protect immunized animals from homologous SHIV 89.6P challenge (35). Furthermore, recent progress in the development of an HIV-1 vaccine expressing multiple Env genes has been made, suggesting the potential production of cross-clade neutralizing antibodies (1, 8, 37, 55). These findings also suggest that the use of sequential immunization with biologically relevant peptides or proteins for the development of HIV-1 vaccines could overcome difficulties associated with otherwise poorly immunogenic epitopes of virus neutralization.

The ability of KD-247 to neutralize HIV-1 may be dependent on site-specific binding to epitopes on the viral envelope glycoprotein. The CDRs of KD-247 were transferred from mouse MAb C25, which was designed to have cross-reactive neutralization activity against HIV-1 clade B isolates. MAb C25 was elicited by sequential immunization using six different peptides containing the V3 GPGR sequence. Antigen recognition by KD-247 was little affected by the genetic reshaping of the C25 gene. Indeed, the results of the Pepscan analysis suggest that KD-247 can react with core V3 sequences from many HIV-1 clade B isolates. The recognition site of KD-247 was mapped to five or six amino acids around the PGR core sequence at the tip of the V3 region of gp120. The shortest peptide that was reactive with KD-247 was regarded as IGPGR, but the epitope is stabilized by the addition of one or more supplemental amino acids. The GPGR sequence of the V3 tip is highly conserved among HIV-1 strains (27, 66). Furthermore, IGPGRA and GPGRAF sequences were detected in the majority of HIV-1 isolates from donors in the United States (34). Using the previously published sequences found in the Los Alamos HIV-1 sequence database, we confirmed that these sequences are present in the majority of HIV-1 clade B isolates (36, 66).

With regard to the properties of the anti-V3 antibody induced by active immunization protocols in animals for HIV vaccine development, the difficulty with exploiting the efficacious antiviral neutralizing antibody responses of anti-V3 antibodies lies in the extraordinary diversity of the V3 sequence and in the resulting strict type specificity of anti-V3 neutralizing antibodies. That strict type specificity is thought to be a function of the inaccessibility of the neutralization epitope on the envelope of the virus (3, 51, 64). This is certainly the case with the polyclonal anti-V3 antibody, which was produced by repeated immunizations with a single HIV Env V3 peptide and which neutralized the only homologous virus in this study. However, sequential immunization of mice with a selection of V3 peptides elicited cross-reactive neutralization antibody responses, and we were eventually able to construct a humanized monoclonal antibody, KD-247, which showed a relatively high level of affinity to the narrow “PGR” motif within the V3 determinant. The humanized antibody was capable of neutralizing a broader range of clade B primary isolates than did the previously reported authentic anti-V3 neutralizing antibody. Thus, the strict type specificity of the anti-V3 antibody elicited by active immunization can be overcome by driving the antibody responses to a more conserved motif within V3. This can be done through sequential immunization with peptides representative of V3 sequences. The KD-247 antibody would be expected to have binding activity against a wide range of HIV-1 field isolates, because the IGPGRA and GPGRAF sequences predominate in the majority of the clade B isolates recognized by the MAb. The results of Pepscan analysis with replacement peptides also suggest that KD-247 has broad binding activity for HIV-1. Although few amino acid substitutions were tolerated in the central PGR sequence of the V3 tip peptide, a number of amino acids substitutions were permitted in the flanking region. It would therefore be expected that KD-247 would bind to HIV-1 quasispecies that have a similar recognition sequence. Interestingly, although the N-NIID isolate showed the same IGPGR V3 tip sequence, it was not neutralized by KD-247 or by any of the other antibodies tested (Table (Table3).3). In contrast, the Pepscan analysis showed that the INIGPGRA V3 tip peptide of the Env region in the N-NIID isolate bound KD-247 using short synthetic peptides (Fig. (Fig.6C,6C, graph b). These results raise the possibility that some amino acids neighboring the neutralization epitope in the viral particle can influence steric hindrance of the binding site, thereby protecting the virus from antibody neutralization.

In studies centered on anti-V3 MAb produced by heterohybridomas using PBMCs from HIV-infected individuals, Gorny et al. recently showed that the V3 loop is accessible on the surface of most primary HIV-1 isolates and serves as a neutralization epitope (23). They also recently identified a quaternary neutralizing epitope on HIV particles (24). Since the fusion of the heteromyeloma and the PBMCs from HIV-infected individuals results in the production of these unique cross-reactive MAbs, a similar sequential immunization with diverse V3 antigens of HIV quasispecies in HIV-infected individuals could lead to the generation of responsible PBMCs, which in turn could produce a cross-neutralizing anti-V3 antibody (21, 23). Actually, human MAb 447-52D shows functional and epitope-mapping characteristics (4, 21) similar to those of the KD-247 humanized antibody described in this study; KD-247 and 447-52D also both possess a similarly narrow binding epitope on the V3 region with high affinity to the “PGR” motif and “GPxR” motif at the center of the V3 region, respectively, and they show cross-reactive neutralization against primary isolates and TCLA isolates. Although KD-247 does not neutralize HIV-1IIIB, which has an insertion of the QR sequence just before the “GPGR” motif in the center of the V3 region, we cannot as of yet compare the breadth of the neutralization activity of the two antibodies, since we used only six primary isolates for this comparison.

The gp120 V1/V2-domain structure and not the sequence variations at the target sites is thought to mediate the neutralization sensitivity of HIV-1 pseudotyped with Env proteins derived from HIV-1 strains SF162 and JR-FL as well as the inherent neutralization resistance of JR-FL and presumably of related primary isolates (48). Despite similar binding affinities for MAbs against V3-, V2- and CD4-binding domains, three MAbs of immunoglobulin G, b12, 2G12, and 2F5, neutralized the JR-FL virus. Using single-cell viral transduction assays mediated by both JR-FL Env pseudotypes and SF162 Env pseudotypes, we plan to compare the neutralization sensitivity of the humanized anti-V3 MAb KD-247 and of immune sera from animals sequentially immunized with a selection of V3 peptides.

The current findings suggest that high affinity for antibody binding is required for neutralization as well as site-specific localization of epitopes to the V3 tip. The kinetic parameters of KD-247 were identified to be fast on rates and slow off rates, similar to those of Rμ5.5, although the KD value of KD-247 for binding to the SP1 peptide was higher than that of Rμ5.5. This higher value results from the faster rate of association for KD-247 (1.3 × 105 M−1 s−1) than that for Rμ5.5 (1.0 × 105 M−1 s−1). This finding is reasonable, since the epitope of KD-247 (IGPGR) is shorter than that of Rμ5.5 (IHIGPGRAFYT). The higher rate of association of KD-247 might be responsible for the virus neutralization activity of the antibody. These results are consistent with hypotheses that propose to use kinetic parameters of antibody binding to explain virus neutralization.

The V3 region of HIV-1 gp120 has been identified as the major determinant of cellular tropism (57) based on coreceptor specificity (63, 65). It is assumed that anti-V3 region neutralizing antibodies inhibit the interaction between the V3 region and its coreceptors. The critical domains, which are concerned with the utilization of the chemokine receptors CCR5 and CXCR4, are located outside of the central IGPGRAF sequence in the V3 region (61). The replacement of amino acids flanking the IGPGRAF sequence had little effect on the binding of KD-247 to peptides, showing that KD-247 neutralized CCR5 and CXCR4 as well as dual-tropic HIV-1 isolates (Fig. 6A and C). In this study, we have shown how neutralizing and nonneutralizing antibodies against primary viruses differ qualitatively. The type-specific anti-V3 MAb Rμ5.5, which binds a linear epitope at the tip of V3 (IHIGPGRAFYT), has the ability to neutralize TCLA CXCR4-tropic virus HIV-1MN but neutralizes neither the CCR5-tropic strain HIV-1AD8 nor the primary virus HIV-1MNp (Fig. (Fig.4).4). In contrast, the humanized anti-V3 antibody KD-247 efficiently neutralized these viruses in neutralization assays using both PBMCs and GHOST cells (Tables (Tables11 and and22).

As previously reported, the envelope spikes of HIV-1 consist of a transmembrane gp41 molecule interacting noncovalently with a gp120 molecule to form an oligomeric structure, most likely a trimer. It has been proposed that the gp120 trimer of TCLA strains forms a relatively open conformation and that the primary isolate trimeric complex has a more closed conformation (4, 49). For these reasons, we propose that a part of the epitope recognized by Rμ5.5 is hidden by the oligomerization of gp120 on primary HIV-1 isolates, reducing its accessibility to the antibody. The ability of KD-247 to neutralize both primary and CCR5-tropic isolates suggests that the V3 tip site protrudes from the gp120 trimer or is located within an area accessible to KD-247 on primary CCR5-tropic isolates but not to Rμ5.5 (Fig. (Fig.7).7). However, Rμ5.5, produced by repeated immunization with SP1, did effectively neutralize homologous TCLA strains of HIV-1 (Fig. (Fig.77).

FIG. 7.
Schematic neutralization model of the cross-reactive anti-V3 MAb KD-247 compared with the type-specific anti-V3 MAb Rμ5.5. Neutralization of primary CCR5-tropic isolates by KD-247 suggests that the narrow V3 tip epitope for KD-247 binding and ...

Our speculation that the V3 tip epitope on the virion is one of the neutralization targets of primary isolates accords well with recent reports suggesting that the V3 epitope is more exposed on intact virions than are the CD4-binding domains and the V2 and gp41 regions (67). Conceivably, nonconformational epitopes may also be responsible for neutralization, since the neutralizing antibody 2F5 recognizes a linear epitope, ELDKWA, in the membrane-proximal region of HIV-1 gp41 (43). Furthermore, HIV-1 envelope proteins have at least three conformational states: an unbound state on the surface of virions, a CD4-bound state, and an end-product state in which the gp120 proteins have dissociated from gp41 trimers (16, 53). Since KD-247 suppresses the ex vivo generation of primary HIV-1 quasispecies in PBMC cultures from HIV-infected individuals (13a), the binding of KD-247 to an appropriate binding epitope on the V3 region of primary HIV-1 can be presumed to occur and to express itself at some point during the three conformational states. KD-247 could then neutralize primary HIV-1.


We thank Richard M. Krause and Malcolm Martin, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md., for their helpful comments.

Valuable reagents and assistance were contributed by Susan Zolla-Pazner with the support of an NIH grant funding the Viral Immunology Core of the NYU Center for AIDS Research (AI 27742). The Panel on AIDS of the US-Japan Cooperative Medical Science Program, the Human Science Foundation, Japan, and the Japanese Ministry of Health, Labor, and Welfare also supported this study.


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