The pharmacokinetics, antigenicity, and fusion-inhibition activity of RSHZ19, a humanized monoclonal antibody to respiratory syncytial virus, in healthy volunteers.

Single ascending doses of RSHZ19 (also known as SB 209763), a humanized monoclonal antibody (MAb) directed to the fusion protein of respiratory syncytial virus, were administered to healthy men to evaluate the safety, pharmacokinetics, antigenicity, and fusion inhibition (FI) activity of RSHZ19. Doses of RSHZ19 (0.025-10.0 mg/kg) or placebo were infused over 30 min, and subjects were followed for 10 weeks. Plasma concentrations of RSHZ19 and RSHZ19-specific antibodies were determined by ELISAs. FI titers were used to evaluate the ability of plasma to inhibit virus-induced fusion of VERO cells previously infected with RS Long strain virus. Twenty-six subjects, mean age 24, completed the study. RSHZ19 was safe and well tolerated, and no subject developed antibodies to RSHZ19 during follow-up. RSHZ19 had low plasma clearance and a half-life of approximately 23 days, similar to native IgG. Increases in FI titers relative to pretreatment levels were seen 24 h after MAb administration in all 4 subjects given 10 mg/kg and in 2 of 4 given 5 mg/kg.

Evaluation of the protective efficacy of reshaped human monoclonal antibody RSHZ19 against respiratory syncytial virus in cotton rats.

Reshaped human MAb RSHZ19, which is specific for the surface fusion protein of respiratory syncytial virus (RSV) is in clinical development for the prevention and treatment of RSV-induced disease in human infants. The current studies profile lung virus clearance and evaluate lung histopathology in MAb-treated, RSV-infected cotton rats, a well characterized model of RSV infection. The highest dose of this MAb (10 mg/kg) administered parenterally 24 h before infection decreased subgroup A or B RSV lung titers to below detectable levels (> or = 2.3 log10 reduction), and significantly reduced lung virus titers (> or = 2.0 log10 reduction) when administered 96 h postinfection. Prophylactic administration of 10 mg/kg RSHZ19 was significantly more protective than 1000 mg/kg conventional human immune serum globulin (HSIg), and protective serum-neutralizing titers in MAb-treated animals (1:32, which correlated with approximately 40 micrograms/ml determined by anti-idiotype ELISA) were significantly lower than those reported previously for HSIg or for convalescent human serum (1:200-1:400). MAb concentration in lung lavages was determined by ELISA to be approximately 1% of the serum MAb concentration, but was not detectable by neutralization assay. The degree of lung histopathology in MAb-treated cotton rats was proportional to lung virus titer, and inversely proportional to the RSHZ19 dose administered. There was no evidence of exacerbated disease in the lungs of MAb-treated animals. These studies thus support the potential clinical utility of RSHZ19 MAb in the prevention and treatment of RSV-induced disease in humans.

Preclinical pharmacokinetic evaluation of the respiratory syncytial virus-specific reshaped human monoclonal antibody RSHZ19.

A preclinical evaluation of RSHZ19, a respiratory syncytial virus-specific reshaped human monoclonal antibody (IgG1 framework), has included pharmacokinetic studies in rats, adult cynomolgus macaques, and infant baboons after intravenous (iv), subcutaneous, or intramuscular (im) administration. After iv administration to rats and monkeys (1 mg/kg dose), a biphasic decline in plasma concentration was observed. The dominant terminal phase was characterized by an 11-day half-life in rats and a 21- to 24-day half-life in monkeys. Plasma clearances of 0.3 ml/hr/kg in the rat and 0.1-0.2 ml/hr/kg in the monkey were estimated. In the macaque, based on area under the curve, no evidence of significant nonlinearity in the pharmacokinetics was observed over a 200-fold dose range (1-200 mg/kg). In rat and monkey, absorption after extravascular administration was rapid relative to elimination (apparent half-lives < or = 24 hr), and bioavailability was high (> or = 82%). After iv or im administration to macaques (> or = 40 mg/kg), 1 of 3 animals in each group developed anti-RSHZ19 antibodies, and this resulted in rapid elimination of RSHZ19 from plasma. After the administration of a second im dose to macaques, no additional animals developed anti-RSHZ19 antibodies. Multiple-dose iv kinetics (5-day repeat dose) in infant baboons were modeled accurately by adult macaque data, suggesting that these species handled RSHZ19 similarly. The pharmacokinetic characteristics of RSHZ19 should support a convenient regimen for treatment or prophylaxis of human respiratory syncytial virus infection.

Influenza A subtype cross-protection after immunization of outbred mice with a purified chimeric NS1/HA2 influenza virus protein.

Influenza A/PR/8/34-derived chimeric (D) protein (SK&F 106160) composed of the first 81 amino acids (aa) of NS1 fused to the conserved 157 C-terminal aa of HA2 (NS1 1-81-HA2 65-222) was previously shown to induce H-2d-restricted protective cytotoxic T-lymphocyte (CTL) immunity in inbred mice. However, D protein, like other small peptides, exhibited haplotype dependence and was not immunogenic in H-2b and H-2K mice. A potential use of this antigen in humans and the role of T cells in any protection were evaluated in outbred Swiss and inbred CBF6F1 (H-2d/b) mice. Mice immunized with D protein and challenged by small-particle aerosol with a lethal dose of influenza virus were significantly protected against mortality from influenza A/H1N1 and A/H2N2 (p < 0.05-< 0.0000001), but not from A/H3N2 and influenza B viruses when compared with control mice. D protein did not induce serum virus-neutralizing antibody but caused virus to be cleared faster in immunized mice. Protection was long-lasting. In vivo depletion of either Lyt2 (CD8+) or L3T4 (CD4+) T cells with monoclonal antibodies led to abrogation of in vitro-generated CTL activity in CF6F1 mice and significant reduction in the protective efficacy of D protein against virus challenge in both Swiss and CF6F1 mice. These results suggest that protection was mediated by CD8+ and/or CD4+ cells and not antibody. Thus D protein, via a conserved sequence on the HA2 polypeptide, has the potential to induce partially cross-reactive CTL that may protect against influenza virus disease in humans.

Induction of protective class I MHC-restricted CTL in mice by a recombinant influenza vaccine in aluminium hydroxide adjuvant.

Induction of class I MHC-restricted cytotoxic T lymphocyte (CTL) responses by soluble proteins or peptides requires complex adjuvants or carrier systems which are not licensed for use with human vaccines. The data presented in this report show that vaccination with a highly purified recombinant influenza protein antigen in aluminium hydroxide adjuvant, the only adjuvant currently licensed for clinical use, elicited class I restricted CTL and protection from lethal challenge with H1N1 and H2N2 viruses. The antigen (D protein, SK&F 106160) is produced by expression of H1N1 influenza virus-derived cDNA (strain A/PR/8/34) in Escherichia coli, and is composed of the first 81 N-terminal amino acids (aa) of the non-structural protein 1 (NS1) fused via a nine nucleotide non-viral linker sequence to the 157 C-terminal aa of the haemagglutinin 2 subunit (HA2). Previous work by Kuwano et al demonstrated that in vitro stimulation of spleen cells from influenza virus-primed mice, with a partially purified preparation of the D protein, selected for CD8+ CTL clones which facilitated lung clearance of H1N1 and H2N2 viruses. In the current study, these results were extended by studying the responses of mice actively immunized with highly purified D protein in the presence or absence of adjuvants. Vaccination of CB6F1 (H-2dxb) mice with D protein in aluminum hydroxide or Freund's complete adjuvant generated H1N1 cross-reactive, H-2d-restricted, CD8+ CTL directed against an immunodominant HA2 epitope (aa 189-199). D protein without adjuvant did not elicit CTL, regardless of the route of injection. However, long-lived (greater than 6 months) splenic memory CTL were elicited by boosting mice intraperitoneally (i.p.) with the D protein in the absence of adjuvant. In mice injected subcutaneously with D protein in aluminium hydroxide at weeks 0 and 3, survival was increased relative to controls up to 16 weeks beyond the second vaccination, after which time additional boosting was required for protection. Studies in H-2b and H-2k mice vaccinated with the D protein showed that induction of CD4+ T-cell or antibody responses, in the absence of CD8+ CTL, did not correlate with protection. Passive transfer of immune sera from CB6F1 mice was also not protective. This prototype H1N1 recombinant subunit vaccine in aluminium adjuvant should directly address the feasibility of achieving a protective cell-mediated immune response in human influenza.