Postinfection passive transfer of KD-247 protects against simian/human immunodeficiency virus-induced CD4+ T-cell loss in macaque lymphoid tissue.

BACKGROUND: Preadministration of high-affinity humanized anti-HIV-1 mAb KD-247 by passive transfer provides sterile protection of monkeys from heterologous chimeric simian/human immunodeficiency virus infection. METHODS: Beginning 1 h, 1 day, or 1 week after simian/human immunodeficiency virus-C2/1 challenge (20 50% tissue culture infective dose), mature, male cynomolgus monkeys received multiple passive transfers of KD-247 (45 mg/kg) on a weekly basis for approximately 2 months. Concentrations and viral loads were measured in peripheral blood, and CD4 T-cell counts were examined in both peripheral blood and various lymphoid tissues. RESULTS: Pharmacokinetic examination revealed similar plasma maintenance levels ranging from 200 to 500 microg/ml of KD-247 in the three groups. One of the six monkeys given KD-247 could not maintain these concentrations, and elicitation of anti-KD-247 idiotype antibody was suggested. All monkeys given KD-247 exhibited striking postinfection protection against both CD4 T-cell loss in various lymphoid tissues and atrophic changes in organs compared with control group animals treated with normal human immunoglobulin G. The KD-247-treated groups were also partially protected against plasma viral load elevation in peripheral blood samples, although the complete protection previously reported with preadministration of this mAb was not achieved. CONCLUSION: Postinfection passive transfer of humanized mAb KD-247 with strong neutralizing capacity against challenged virus simian/human immunodeficiency virus-C2/1 protected CD4 T cells in lymphoid organs.

Anti-V3 humanized antibody KD-247 effectively suppresses ex vivo generation of human immunodeficiency virus type 1 and affords sterile protection of monkeys against a heterologous simian/human immunodeficiency virus infection.

In an accompanying report (Y. Eda, M. Takizawa, T. Murakami, H. Maeda, K. Kimachi, H. Yonemura, S. Koyanagi, K. Shiosaki, H. Higuchi, K. Makizumi, T. Nakashima, K. Osatomi, S. Tokiyoshi, S. Matsushita, N. Yamamoto, and M. Honda, J. Virol. 80:5552-5562, 2006), we discuss our production of a high-affinity humanized monoclonal antibody, KD-247, by sequential immunization with V3 peptides derived from human immunodeficiency virus type 1 (HIV-1) clade B primary isolates. Epitope mapping revealed that KD-247 recognized the Pro-Gly-Arg V3 tip sequence conserved in HIV-1 clade B isolates. In this study, we further demonstrate that in vitro, KD-247 efficiently neutralizes CXCR4- and CCR5-tropic primary HIV-1 clade B and clade B' with matching neutralization sequence motifs but does not neutralize sequence-mismatched clade B and clade E isolates. Monkeys were provided sterile protection against heterologous simian/human immunodeficiency virus challenge by the passive transfer of a single high dose (45 mg per kg of body weight) of KD-247 and afforded partial protection by lower antibody doses (30 and 15 mg per kg). Protective neutralization endpoint titers in plasma at the time of virus challenge were 1:160 in animals passively transferred with a high dose of the antibody. The antiviral efficacy of the antibody was further confirmed by its suppression of the ex vivo generation of primary HIV-1 quasispecies in peripheral blood mononuclear cell cultures from HIV-infected individuals. Therefore, KD-247 promises to be a valuable tool not only as a passive immunization antibody for the prevention of HIV infection but also as an immunotherapy for the suppression of HIV in phenotype-matched HIV-infected individuals.

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.

Ex vivo neutralization of HIV-1 quasi-species by a broadly reactive humanized monoclonal antibody KD-247.

By immunizing mice sequentially with six different V3 peptides we obtained a murine monoclonal antibody (MAb) C25, and its humanized counterpart KD-247. The MAb recognizes the sequence IGPGRA at the tip of the V3 loop and displays broad neutralizing activity against a variety of HIV-1 isolates. KD-247 was tested in an ex vivo neutralization assay to determine its capability to contain the spread of a quasi species population of clade B HIV-1 derived from two patients. The epitope of KD-247 was generally matching with the V3 sequences of various clones isolated from plasma and peripheral blood mononuclear cells (PBMC) of two patients. Complete or strong inhibition of viral replication was observed when the patients' PBMC were cultured with a high concentration of KD-247. Some neutralization escape variants, which had mutations in the V3 or outside of the V3 loop, emerged only at a low concentration of the MAb. These results suggest that KD-247 could be a good candidate for immunotherapy against HIV-1 in vivo.

Hemagglutinating virus of Japan protein is efficient for induction of CD4+ T-cell response by a hepatitis B core particle-based HIV vaccine.

By using the hepatitis B core (HBc) protein gene as a carrier, HIV-1 env V3 gene was inserted into the carrier gene, and the HIV gene was expressed inside a chimeric HIV-HBc particle (HIV-HBc), which was a unique candidate for induction of HIV-specific CTL activity. This was seen significantly in mice without the need of an adjuvant, because other responses specific for the HIV peptide such as T-cell proliferation and antibody production were not induced. However, when hemagglutinating virus of Japan (HVJ) protein was incorporated into an anionic liposome containing HIV peptide (HIV-HVJ-liposome) and was used as a booster immunization in HIV-HBc primed animals, the HIV-specific T-cell response and enhanced CTL activity were clearly induced in consecutively immunized animals. Furthermore, the HIV-specific humoral immune response was also induced and a neutralization activity was detected in the immune sera. Thus, when an HIV peptide antigen is expressed inside the virus like a particle of HBc, it can induce both cellular and humoral immunities when an HVJ-HIV-liposome, but not an HIV-liposome, is inoculated as the booster antigen. The HVJ-stimulated splenocytes secreted IL-18 and IL-12 to synergistically enhance the secretion of IFN-gamma in vitro. These findings suggest that the HVJ protein is effective at inducing the HIV-specific immunities, if used as part of a booster antigen in the consecutive immunization regimen.