Sachio Tokiyoshi

Construction of reshaped human antibodies with HIV-neutralizing activity

Mouse monoclonal antibody (mAb) 0.5 beta binds to the envelope protein gp120 of human immunodeficiency virus (HIV) and neutralizes infection by HIV in vitro. Mouse mAb 0.5 beta, therefore, has potential as a therapeutic agent for the prevention and treatment of acquired immunodeficiency syndrome (AIDS). Since mouse mAbs are highly immunogenic in humans, efforts are being made to humanize mouse mAbs that are being considered for use in humans. This report describes the design, construction, and expression of reshaped human 0.5 beta antibodies. In these antibodies, the entire constant (C) regions were derived from human sequences. The variable (V) regions were derived from human framework regions (FRs) and mouse 0.5 beta complementarity determining regions (CDRs). One version of reshaped human 0.5 beta light (L) chain and six versions of reshaped human 0.5 beta heavy (H) chain were made and tested. Following transient expression in cos cells, all of the constructions were capable of producing humanlike antibody. Three of the H chain constructions (RHc, RHe, and RHf), when co-expressed with the L chain construction (RL), produced reshaped human antibody capable of binding to the epitope on gp120 recognized by mouse 0.5 beta mAb. The best version (RL + RHe) of reshaped human 0.5 beta antibody had both binding affinity and neutralizing activity that were within twofold that of the mouse or chimeric 0.5 beta antibody.

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.

Microencapsulation of Hepatitis B Core Antigen for Vaccine Preparation

AbstractPurpose. To prepare poly(lactide-co-glycolide)(PLGA) microspheres containing recombinant hepatitis B core antigen (HBcAg; Mw = 3,600,000) by a w/o/w emulsion/solvent evaporation method and evaluate the possibility of this system as a potent long-acting carrier for hepatitis B core antigen in mice. Methods. Various additives had been incorporated in the internal aqueous phase during the process of microencapsulating HBcAg, HBcAg antigenicity in the medium extracted from the prepared microspheres were measured by ELISA. Shape confirmation of the HBcAg antigen was performed by a sucrose gradient velocity centrifugal technique. For in vivo study, prepared microspheres were administered subcutaneously to Balb/C mice, and the serum IgG level was determined by ELISA. Results. The inactivation of HBcAg by methylene chloride was dramatically reduced by the addition of gelatin (4−8% (w/v)) to the internal aqueous phase during the preparation. Further improvement of the loading efficiency to almost 61% resulted with cooling (4°C). The prepared microspheres (4.27 μm ± 1.23 μm) containing 0.15% HBcAg displayed burst release (50−60% within 2 days). In subcutaneous inoculation, the adjuvant effect of PLGA microspheres was almost the same as that of the complete Freunds adjuvant. Whereas oral inoculation using the microspheres was not effective. Conclusions. The pH of the added gelatin seemed to be the key to the stabilization of HBcAg from various stability tests and CD spectrum study. Finally, the possibility of using this system as a potent long-acting hepatitis B vaccine was demonostrated.

Sendai virus-based production of HIV type 1 subtype B and subtype E envelope glycoprotein 120 antigens and their use for highly sensitive detection of subtype-specific serum antibodies.

We previously described a Sendai virus (SeV)-based expression system for the recombinant gp120 of HIV-1 subtype B (rgp120-B), which has permitted the production of antigenetically and functionally authentic gp120 at a concentration as high as 6 microg/ml of culture supernatant (Yu D et al.: Genes Cells 1997;2:457-466). Here the same procedure was successfully applied to the production of HIV-1 subtype E gp120 (rgp120-E). The remarkable production of the proteins by the SeV expression system enabled us to use crude culture supernatants for serological and functional studies of gp120s. The immunological authenticity of rgp120-E was verified by patient sera and anti-V3 loop monoclonal antibodies specific for HIV-1 subtypes B and E. CD4-binding properties were corroborated by FACS analyses. The rgp120s were then used in an enzyme immunoassay (rgp120-EIA) to detect antibodies in the sera of HIV-1-infected individuals, and the performance was assessed in comparison with a conventional V3 loop peptide EIA (V3-EIA). The initial evaluation of a serum panel (n = 164) consisting of 76 subtype E and 88 subtype B sera revealed that the rgp120-EIA was nearly 1000-fold more sensitive than the V3-EIA and was able to detect subtype-specific antibody with 100% sensitivity and with a complete correlation with the genotypes, whereas the V3-EIA failed to detect 9 and 24% of the same subtype E and B sera, respectively. Furthermore, a study employing a panel of 28 international sera with known genotypes (HIV-1 subtypes A through F) confirmed the remarkable specificity of this method. An EIA reactivity higher than 1.0 was an unambiguous predictor of HIV-1 subtype E and B infections. The data imply the presence of strong subtype-specific epitopes for antibody bindings to these rgp120s.

Father-to-Mother-to-Infant Transmission of HIV-1:Clonally Transmitted Isolate of Infant Mutates More Rapidly than That of the Mother and Rapidly Loses Reactivity with Neutralizing Antibody

The sequences of the V3 loop and surrounding regions of human immunodeficiency virus type‐1 from a father‐to‐mother‐to‐infant trimmer were studied and the horizontal and vertical transmissions compared. The fathers virus was variable for reactivity with neutralizing antibody and sequences of the V3 loop central core sequence. In contrast, the mothers viral sequences were much less diverse and reacted with a virus neutralizing antibody. The infants viral sequences were also less diverse than those of the father, and N‐glycosylation sites were conserved. By phylogenetic analysis, the major clone, of which V3‐peptide reacted with the neutralizing antibody, was found to be transmitted from the mother to her infant; however, the mutated minor clones did not bind to the antibody. These findings suggest that both horizontal and vertical virus transmission were selective, and that the clonally transmitted virus in infants mutates more rapidly than viruses in the mother, to whom the virus was horizontally transmitted.