Mari Takizawa

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

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.

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.

ABSTRACT 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.

Suppressive Mechanisms of EPA on Human T Cell Proliferation

In vivo and in vitro experiments show that polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) inhibit mitogen‐ or antigen‐stimulated proliferation of T cells in rodents and humans. However, the exact manner and mechanisms by which PUFA inhibits T cell proliferation is not clear. In the present study, we investigated the suppressive effects of EPA, an n‐3 PUFA, on PHA stimulated human peripheral blood T cells. Our results showed that EPA suppresses mitogen‐ or antigen‐stimulated human T cell proliferation by at least 2 steps; step 1) EPA suppresses T cell proliferation by inhibiting IL‐2Rα expression and IL‐2 production; step 2) EPA induces cell death of blast T cells without reducing the expression of IL‐2Rα. We also showed that EPA selectively stimulates the cell death of blast T cells but not resting T cells. The suppressive effect of EPA was mediated via the production of reactive oxygen products, because EPA‐stimulated H2O2 production and the suppressive effect of EPA was restored by addition of catalase or NAC. These results taken together suggest that such immunosuppressive effects of EPA may explain the apparent benefits of EPA‐enriched diets for patients with inflammatory disorders.

Presence of multiple HIV type 1 subtypes among mothers and children in Japan.

We collected blood samples from 70 HIV-1-infected pregnant women and 76 babies born to HIV-1-infected women in Japan, from 1989 to 1999. To analyze the genetic diversity of HIV-1 among mothers and children, we sequenced the C2-V3 regions of HIV-1 gp120. Phylogenetic tree analysis of these regions revealed that multiple HIV-1 subtypes, A, B, D, E, and G, were circulating among mothers and children in Japan. Thus, the genetic heterogeneity of HIV-1 among mothers and children in Japan is steadily increasing, although the number of cases remains small. Perhaps the longest term survivor, an 11-year-old child with a vertical HIV-1 subtype G infection in Japan, is one of our subjects.

Therapeutic potential of HIV protease-activable CASP3

Development of a therapeutic application of CASP3/caspase 3/CPP32, an executor of apoptosis, has been challenging because regulation of its activation is complicated. This study aimed to inhibit cancer cell growth and human immunodeficiency virus type 1 (HIV-1) propagation through a CASP3 mutant, CASP3*, activable by HIV-1-encoded aspartate protease. Active CASP3* was delivered to leukemic cells using a protein transduction vehicle, the lentivirus-like nanoparticle (LENA), which should contain thousands of CASP3*-Gag protein molecules and release the activated CASP3* into the target cell cytoplasm. CASP3*-LENA induced apoptosis in various types of leukemic cells. In addition to being effective against leukemic cells, constitutive expression of CASP3* restricted HIV-1 propagation in SUP-T1 cells. The attenuation of HIV-1 replication in SUP-T1/CASP3* cells was attributed to the elimination of HIV-1-infected cells by apoptosis. These data suggest that CASP3* has therapeutic potential against both lymphoid malignancies and HIV-1 infection.

Eicosapentaenoic acid inhibits CSF‐induced human monocyte survival and maturation into macrophage through the stimulation of H2O2 production

Human studies suggest a beneficial effect of eicosapentaenoic acid (EPA)‐supplemented diets on atherosclerotic and atherothrombotic disorders as well as autoimmune and inflammatory diseases and tumors. The effects of EPA on human monocyte survival and maturation into macrophage are not yet known. We studied the effects of EPA on the survival and development into macrophage of human monocyte treated with colony‐stimulating factor (CSF). We have found that EPA induces cell death of the monocyte via apoptosis, even in the presence of M‐CSF or GM‐CSF, and inhibits differentiation from the monocyte to macrophage by inducing H2O2 production. In contrast to the effect of EPA on monocytes, EPA did not induce cell death of monocyte‐derived macrophages. Such an apoptosis inducing effect on monocytes by EPA may contribute to the efficacy of EPA in atherosclerosis and autoimmune diseases.

Regulation of the susceptibility of HIV-1 to a neutralizing antibody KD-247 by nonepitope mutations distant from its epitope.

Objective:A humanized neutralizing antibody, KD-247, targets the V3 loop of HIV-1 Env. HIV-1 bearing the GPGR sequence at the V3 loop is potentially susceptible to KD-247. However, not all GPGR-positive HIV-1 isolates are neutralized by KD-247. We examined the potential mechanism by which the susceptibility of HIV-1 to KD-247-mediated neutralization is regulated. Design:We searched for nonepitope neutralization regulatory (NNR) mutations that sensitize GPGR-bearing HIV-1AD8 to KD-247 and mapped the locations of such mutations relative to the V3 loop. Methods:We generated a functional HIV-1AD8 Env library, and evaluated the viral susceptibility to KD-247 by measuring the half-inhibitory concentration (IC50) to KD-247 on TZM-bl cell assay. Results:We identified nine KD-247-sensitizing NNR mutations from 30 mutations in various regions of gp120, including the V1/V2 loop, C2, V3 loop, C4, and C5. They specifically affected KD-247-mediated neutralization, as they did not affect the b12-mediated neutralization. When combined, the KD-247-sensitizing NNR mutations additively sensitized the virus to KD-247 by up to 10 000 folds. The KD-247-sensitizing NNR mutations increased KD-247 binding to the virion. Notably, the NNR mutation in C4 coincides with the CD4-binding site of gp120. Conclusion:Given that most of the KD-247-sensitizing NNR mutations are remote from V3 loop, it is reasonable to hypothesize that the steady-state, local conformation of the V3 loop is regulated by the interdomain contact of gp120. Our mutational analysis complements crystallographic studies by helping provide a better understanding of the steady-state conformation and the functional geometry of Env.

Decline in the HIV-1 isolation rate in Japan: a 12-year observation.

Since 1988, we have isolated HIV‐1 from 614 HIV‐1‐infected persons (total sample = 2,785) in Japan. During the past 12 years, we have found a decline in the HIV‐1 isolation rate in Japan, with two identifiable turning points, 1991–1992 and 1996–1997. The two turning points correspond to shifts in anti‐HIV‐1 therapy. These findings suggest that HIV‐1 in Japan is currently biologically well controlled, probably due to anti‐HIV‐1 therapy. On the other hand, this decline is inconsistent with the recent increase of genetic drug‐resistant HIV‐1 in Japan. Further studies are needed to clarify mechanisms that might explain the discrepancy.

Conformational properties of the third variable loop of HIV-1AD8 envelope glycoprotein in the liganded conditions

The conformational dynamics of the HIV-1 envelope glycoprotein gp120 and gp41 (Env) remains poorly understood. Here we examined how the V3 loop conformation is regulated in the liganded state using a panel of recombinant HIV-1NL4-3 clones bearing HIV-1AD8 Env by two experimental approaches, one adopting a monoclonal neutralizing antibody KD-247 (suvizumab) that recognizes the tip of the V3 loop, and the other assessing the function of the V3 loop. A significant positive correlation of the Env-KD-247 binding was detected between the liganded and unliganded conditions. Namely, the mutation D163G located in the V2 loop, which enhances viral susceptibility to KD-247 by 59.4-fold, had little effect on the sCD4-induced increment of the virus-KD-247 binding. By contrast, a virus with the S370N mutation in the C3 region increased the virus-KD-247 binding by 91.4-fold, although it did not influence the KD-247-mediated neutralization. Co-receptor usage and the susceptibility to CCR5 inhibitor Maraviroc were unaffected by D163G and S370N mutations. Collectively, these data suggest that the conformation of the liganded V3-loop of HIV-1AD8 Env is still under regulation of other Env domains aside from the V3 loop, including V2 and C3. Our results give an insight into the structural properties of HIV-1 Env and viral resistance to entry inhibitors by non-V3 loop mutations.