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. 2006 Jun;80(11):5552-62.
doi: 10.1128/JVI.02094-05.

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

Yasuyuki Eda  1 , Mari Takizawa, Toshio Murakami, Hiroaki Maeda, Kazuhiko Kimachi, Hiroshi Yonemura, Satoshi Koyanagi, Kouichi Shiosaki, Hirofumi Higuchi, Keiichi Makizumi, Toshihiro Nakashima, Kiyoshi Osatomi, Sachio Tokiyoshi, Shuzo Matsushita, Naoki Yamamoto, Mitsuo Honda
Affiliations

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

Yasuyuki Eda et al. J Virol. 2006 Jun.

Abstract

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.

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Figures

FIG. 1.
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 antisera from an animal sequentially immunized with the V3 peptides. (C) Binding antibody titers of antisera from animals immunized six times with SP1 alone. (D) Binding antibody titers of an isolated C25 MAb against various V3 peptides. Binding antibody titers were similarly determined by monitoring the antibody reactivity in a peptide-based ELISA. The coated peptides were SP1 (•), SP17 (○), SP11 (▴), SP14 (▵), SP12 (▪), SP13 (□), SP6 (⧫), SP9 (⋄), and SP10 (×).
FIG. 2.
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 of HIV-1 strains MN (A), MNp (B), and JR-CSF (C) by immune sera from sequentially immunized mice with diverse V3 tip peptides (▪) or by immune sera from mice with repeated immunization with a single SP1 peptide (▴) determined by a PBMC-based virus neutralization assay.
FIG. 3.
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 human V regions, which were transferred from the mouse MAb. Dashes indicate a deletion. The sequence of a control reshaping MAb, Rμ5.5, derived from a murine MAb obtained by immunization with SP1 peptide alone is also shown.
FIG. 4.
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 for the neutralization assay; results are expressed as the means of data from four different assays of triplicate samples.
FIG. 5.
FIG. 5.
Sensorgram overlays for the binding of KD-247, Rμ5.5, and Cβ1 to the SP1 peptide.
FIG. 6.
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 any deletion (A) or replacement (C). The amino acid deleted from the original sequence is indicated on the horizontal axis (A). The original sequences for the set of deletion analogues were IHIGPGRAFY (A, panel a), RVGPGRTL (A, panel b), RVGPGRAI (A, panel c), and SVGPGRSF (A, panel d), derived from HIV-1 strains MN, NI53, NI54, and TM2, respectively. The decamer peptide derived from the V3 region of the MN strain was also synthesized; overlapping peptides of multiple lengths (B) and replacement peptides (C) are shown. The length of each set of peptides and the sequences, which exhibited relatively strong binding activity, are indicated on the vertical axis (B). Every group of 20 lines corresponds to the complete replacement set for 1 of the 10 amino acid positions in the decamer peptide (C, panels a to j). The amino acid replaced with the original sequence is indicated on the horizontal axis (c).
FIG. 7.
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 neutralization sticks out from the gp120 trimer of primary CCR5-tropic isolates, or it is located in an accessible site of the gp120 trimer during neutralization by KD-247. However, Rμ5.5 may not be able to bind to the V3 tip epitope, since the neutralization epitope for Rμ5.5 is broader than KD-247's epitope (Fig. 6), which may not be fully expressed and are more efficiently masked in the primary R5-tropic viruses compared to TCLA strains.
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