An anti-H5N1 influenza virus FcDART antibody is a highly efficacious therapeutic agent and prophylactic against H5N1 influenza virus infection.

UNLABELLED: Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. While vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic agent that is highly efficacious at low doses and is broadly specific against antigenically drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific Fc fusion protein (the Fc dual-affinity retargeting [FcDART] molecule). In mice, a single therapeutic or prophylactic dose of either monoclonal antibody at 2.5 mg/kg of body weight provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1-mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5-mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, on the basis of analysis of mutants with antibody escape mutations. While it was possible to generate escape mutants in vitro, they were neutralized by the antibodies in vivo, as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutant in vitro. In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART molecule, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses. IMPORTANCE: Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and are a pandemic threat. A vaccine is available, but other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. The variability of the virus means that such an approach must be broad spectrum. To achieve this, we developed two antibodies that neutralize H5N1 influenza viruses. In mice, these antibodies provided complete protection against a spectrum of H5N1 influenza viruses at a single low dose. We then combined the two antibodies into a single molecule, FcDART, which combined the broad-spectrum activity and protective efficacy of both antibodies. This treatment provides a novel and effective therapeutic agent or prophylactic with activity against highly pathogenic H5N1 avian influenza viruses.

Analysis of STAT laboratory turnaround times before and after conversion of the hospital information system.

BACKGROUND: Modern electronic health record systems (EHRS) reportedly offer advantages including improved quality, error prevention, cost reduction, and increased efficiency. This project reviewed the impact on specimen turnaround times (TAT) and percent compliance for specimens processed in a STAT laboratory after implementation of an upgraded EHRS. METHODS: Before EHRS implementation, laboratory personnel received instruction and training for specimen processing. One laboratory member per shift received additional training. TAT and percent compliance data sampling occurred 4 times monthly for 13 months post-conversion and were compared with the mean of data collected for 3 months pre-conversion. Percent compliance was gauged using a benchmark of reporting 95% of all specimens within 7 min from receipt. RESULTS: Control charts were constructed for TAT and percent compliance with control limits set at 2 SD and applied continuously through the data collection period. TAT recovered to pre-conversion levels by the 6th month post-conversion. Percent compliance consistently returned to pre-conversion levels by the 10th month post-conversion. Statistical analyses revealed the TAT were significantly longer for 3 months post-conversion (P < .001) compared with pre-conversion levels. Statistical significance was not observed for subsequent groups. Percent compliance results were significantly lower for 6 months post-conversion (P < .001). Statistical significance was not observed for subsequent groups. CONCLUSIONS: Extensive efforts were made to train and prepare personnel for challenges expected after the EHRS upgrade. Specific causes identified with the upgraded EHRS included multiple issues involving personnel and the EHRS. These data suggest that system and user issues contributed to delays in returning to pre-conversion TAT and percent compliance levels following the upgrade in the EHRS.

PCPP-formulated H5N1 influenza vaccine displays improved stability and dose-sparing effect in lethal challenge studies.

The potential impact of an influenza pandemic can be mitigated through the realization of a successful vaccination program. The implementation of antigen stabilization and dose-sparing technologies is an important step in improving availability of vaccines at the time of a pandemic outbreak. We investigated poly[di(carboxylatophenoxy)phosphazene] (PCPP) as a potential stabilizing and immunostimulating agent for H5N1 influenza vaccine. Physicochemical characterization of PCPP-formulated H5N1 influenza vaccine revealed macromolecular complexation in the system, whereas single radial immunodiffusion assay verified antigenicity of the formulation in vitro. PCPP-enhanced formulation displayed a fourfold increase in the half-life at 40°C compared with a nonadjuvanted vaccine. Lethal challenge studies in ferrets demonstrated 100% protection for low-antigen dose PCPP-adjuvanted formulations (1 µg of hemagglutinin) and at least a 10-fold antigen-sparing effect. Therefore, PCPP demonstrated an ability to improve thermal stability of H5N1 influenza vaccine in solutions and provide for a substantial dose-sparing effect in vivo.

Feasibility of reconstructed ancestral H5N1 influenza viruses for cross-clade protective vaccine development.

Since the reemergence of highly pathogenic H5N1 influenza viruses in humans in 2003, these viruses have spread throughout avian species in Asia, Europe, and Africa. Their sustained circulation has resulted in the evolution of phylogenetically diverse lineages. Viruses from these lineages show considerable antigenic variation, which has confounded vaccine planning efforts. We reconstructed ancestral protein sequences at several nodes of the hemagglutinin (HA) and neuraminidase (NA) gene phylogenies that represent ancestors to diverse H5N1 virus clades. By using the same methods that have been used to generate currently licensed inactivated H5N1 vaccines, we were able to produce a panel of replication competent influenza viruses containing synthesized HA and NA genes representing the reconstructed ancestral proteins. We identified two of these viruses that showed promising in vitro cross-reactivity with clade 1, 2.1, 2.2, 2.3.4, and 4 viruses. To confirm that vaccine antigens derived from these viruses were able to elicit functional antibodies following immunization, we created whole-virus vaccines and compared their protective efficacy versus that of antigens from positive control, naturally occurring, and broadly reactive H5N1 viruses. The ancestral viruses' vaccines provided robust protection against morbidity and mortality in ferrets challenged with H5N1 strains from clades 1, 2.1, and 2.2 in a manner similar to those based on the control strains. These findings provide proof of principle that viable, computationally derived vaccine seed viruses can be constructed within the context of currently licensed vaccine platforms. Such technologies should be explored to enhance the cross reactivity and availability of H5N1 influenza vaccines.

Inactivated seasonal influenza vaccines increase serum antibodies to the neuraminidase of pandemic influenza A(H1N1) 2009 virus in an age-dependent manner.

Levels of preexisting antibodies to the hemagglutinin of pandemic influenza A(H1N1) 2009 (hereafter pandemic H1N1) virus positively correlate with age. The impact of contemporary seasonal influenza vaccines on establishing immunity to other pandemic H1N1 proteins is unknown. We measured serum antibodies to the neuraminidase (NA) of pandemic H1N1 in adults prior to and after vaccination with seasonal trivalent inactivated influenza vaccines. Serum antibodies to pandemic H1N1 NA were observed in all age groups; however, vaccination elevated levels of pandemic H1N1 NA antibodies predominately in elderly individuals (age, ⩾60 years). Therefore, contemporary seasonal vaccines likely contribute to reduction of pandemic H1N1-associated disease in older individuals.

Puzzling inefficiency of H5N1 influenza vaccines in Egyptian poultry.

In Egypt, efforts to control highly pathogenic H5N1 avian influenza virus in poultry and in humans have failed despite increased biosecurity, quarantine, and vaccination at poultry farms. The ongoing circulation of HP H5N1 avian influenza in Egypt has caused >100 human infections and remains an unresolved threat to veterinary and public health. Here, we describe that the failure of commercially available H5 poultry vaccines in Egypt may be caused in part by the passive transfer of maternal H5N1 antibodies to chicks, inhibiting their immune response to vaccination. We propose that the induction of a protective immune response to H5N1 is suppressed for an extended period in young chickens. This issue, among others, must be resolved and additional steps must be taken before the outbreaks in Egypt can be controlled.