Efficacy of the novel parainfluenza virus haemagglutinin-neuraminidase inhibitor BCX 2798 in mice - further evaluation.

BACKGROUND: Human parainfluenza virus type 1 (hPIV-1) causes serious respiratory tract infections, especially in children. This study investigated the efficacy of the novel haemagglutinin-neuraminidase (HN) inhibitor BCX 2798 in the prophylaxis of lethal and the treatment of non-lethal parainfluenza virus infection in mice. METHODS: In the prophylaxis model, 129x1/SvJ mice were inoculated with a 90% lethal dose of a recombinant Sendai virus, in which the HN gene was replaced with that of hPIV-1 (rSeV[hPIV-1HN]). The mice were intranasally treated either once or for 5 days with 1 or 10 mg/kg/day of BCX 2798, starting 4 h before infection. In the therapeutic model, mice were infected with 100 plaque-forming units of rSeV(hPIV-1HN) per mouse and treated intranasally with 0.1, 1 or 10 mg/kg/day of BCX 2798 for 5 days, starting 24 or 48 h after infection, or for 4 days starting 72 h after infection. RESULTS: Similar to multiple dosing, a single intranasal prophylaxis with 1 or 10 mg/kg of BCX 2798 protected approximately 40% or 90%, respectively, of mice from death by rSeV(hPIV-1HN) infection. BCX 2798 also significantly reduced virus lung titres (in a dose- and time-dependent manner) and reduced histopathological changes in the airways of non-lethally infected mice at multiple intranasal dosages in the therapeutic model, with the lowest effective dosage being 0.1 mg/kg/day administered 24 h after infection. CONCLUSIONS: BCX 2798 was effective in the prophylaxis of lethal and in the therapy of non-lethal parainfluenza virus infection in mice, suggesting further consideration of BCX 2798 for clinical trials.

Effect of hemagglutinin-neuraminidase inhibitors BCX 2798 and BCX 2855 on growth and pathogenicity of Sendai/human parainfluenza type 3 chimera virus in mice.

Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects young children, but no vaccines or antiviral drugs against it have yet been developed. We developed a mouse model to evaluate the efficacies of the novel parainfluenza virus hemagglutinin-neuraminidase (HN) inhibitors BCX 2798 and BCX 2855 against a recombinant Sendai virus (rSeV) in which the fusion (F) and HN surface glycoproteins (FHN) were replaced by those of hPIV-3 [rSeV(hPIV-3FHN)]. In the prophylaxis model, 129X1/SvJ mice were infected with a 90% or 20% lethal dose of the virus and were treated intranasally for 5 days with 10 mg/kg of body weight/day of either compound starting 4 h before infection. Prophylactic treatment of the mice with either compound did not prevent their death in a 90% lethality model of rSeV(hPIV-3FHN) infection. However, it significantly reduced the lung virus titers, the amount of weight lost, and the rate of mortality in mice infected with a 20% lethal virus dose. In the therapy model, mice were infected with a nonlethal dose of the virus (100 PFU/mouse) and were treated intranasally with 1 or 10 mg/kg/day of either compound for 5 days starting at 24 or 48 h postinfection. Treatment of the mice with either compound significantly reduced the virus titer in the lungs, subsequently causing a reduction in the number of immune cells and the levels of cytokines in the bronchoalveolar lavage fluid and histopathologic changes in the airways. Our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of hPIV-3 HN in our mouse model and may be promising candidates for the prophylaxis and treatment of hPIV-3 infection in humans.

Loss of the N-linked glycan at residue 173 of human parainfluenza virus type 1 hemagglutinin-neuraminidase exposes a second receptor-binding site.

BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

The novel parainfluenza virus hemagglutinin-neuraminidase inhibitor BCX 2798 prevents lethal synergism between a paramyxovirus and Streptococcus pneumoniae.

An association exists between respiratory viruses and bacterial infections. Prevention or treatment of the preceding viral infection is a logical goal for reducing this important cause of morbidity and mortality. The ability of the novel, selective parainfluenza virus hemagglutinin-neuraminidase inhibitor BCX 2798 to prevent the synergism between a paramyxovirus and Streptococcus pneumoniae was examined in this study. A model of secondary bacterial pneumonia after infection with a recombinant Sendai virus whose hemagglutinin-neuraminidase gene was replaced with that of human parainfluenza virus type 1 [rSV(hHN)] was established in mice. Challenge of mice with a sublethal dose of S. pneumoniae 7 days after a sublethal infection with rSV(hHN) (synergistic group) caused 100% mortality. Bacterial infection preceding viral infection had no effect on survival. The mean bacterial titers in the synergistic group were significantly higher than in mice infected with bacteria only. The virus titers were similar in mice infected with rSV(hHN) alone and in dually infected mice. Intranasal administration of BCX 2798 at 10 mg/kg per day to the synergistic group of mice starting 4 h before virus infection protected 80% of animals from death. This effect was accompanied by a significant reduction in lung viral and bacterial titers. Treatment of mice 24 h after the rSV(hHN) infection showed no protection against synergistic lethality. Together, our results indicate that parainfluenza viruses can prime for secondary bacterial infections. Prophylaxis of parainfluenza virus infections with antivirals might be an effective strategy for prevention of secondary bacterial complications in humans.

Efficacy of novel hemagglutinin-neuraminidase inhibitors BCX 2798 and BCX 2855 against human parainfluenza viruses in vitro and in vivo.

Human parainfluenza viruses are important respiratory tract pathogens, especially of children. However, no vaccines or specific therapies for infections caused by these viruses are currently available. In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein. The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK(2) cells. The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 micro M in HA inhibition assays and from 0.02 to 20 micro M in NA inhibition assays. The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 micro M. BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA. In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death. Treatment beginning 24 h after the start of infection did not prevent death. Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans.