Potency Testing of Venoms and Antivenoms in Embryonated Eggs: An Ethical Alternative to Animal Testing.

Venoms are complex mixtures of biologically active molecules that impact multiple physiological systems. Manufacture of antivenoms (AVs) therefore requires potency testing using in vivo models to ensure AV efficacy. As part of ongoing research to replace small animals as the standard model for AV potency testing, we developed an alternate in vivo method using the embryonated egg model (EEM). In this model, the survival of chicken embryos envenomated in ovo is determined prior to 50% gestation, when they are recognized as animals by animal welfare legislation. Embryos were found to be susceptible to a range of snake, spider, and marine venoms. This included funnel-web spider venom for which the only other vertebrate, non-primate animal model is newborn mice. Neutralization of venom with standard AV allowed correlation of AV potency results from the EEM to results from animal assays. Our findings indicate that the EEM provides an alternative, insensate in vivo model for the assessment of AV potency. The EEM may enable reduction or replacement of the use of small animals, as longer-term research that enables the elimination of animal use in potency testing continues.

The source of the PB1 gene in influenza vaccine reassortants selectively alters the hemagglutinin content of the resulting seed virus.

The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield.

Development of an enzyme-linked immunoassay for the quantitation of influenza haemagglutinin: an alternative method to single radial immunodiffusion.

BACKGROUND: The current method used to measure haemagglutinin (HA) content for influenza vaccine formulation, single radial immunodiffusion (SRID), is lengthy and relies on the availability of matched standardised homologous reagents. The 2009 influenza pandemic highlighted the need to develop alternate assays that are able to rapidly quantitate HA antigen for vaccine formulation. OBJECTIVES: The aim of this work was to develop an enzyme-linked immunoassay (EIA) for the rapid quantitation of H1, H3, H5 and B influenza HA antigens. METHODS: Monoclonal antibodies (mAbs) selected for haemagglutination inhibition (HAI) activity were conjugated with horseradish peroxidase and used to establish a capture-detection EIA for the quantitation of HA antigen. Results were compared with the appropriate reference SRID assays to investigate assay performance and utility. RESULTS: Quantitation of HA antigen by EIA correlated well with current reference SRID assays. EIA results showed equivalent precision and exhibited a similar capacity to detect HA antigen in virus samples that had been used in either stability or splitting studies, or subjected to physical or chemical stresses. EIA exhibited greater sensitivity than SRID and has the potential to be used in high-throughput applications. CONCLUSIONS: We demonstrated the utility of EIA as a suitable alternative to SRID for HA antigen quantitation and stability assessment. This approach would lead to earlier availability of both seasonal and pandemic vaccines, because of the extended cross-reactivity of reagents.

Abortive replication of influenza virus in mouse dendritic cells.

The interaction between influenza virus and dendritic cells (DCs) remains poorly defined and controversial. Here we show that influenza virus replication in mouse bone marrow-derived DCs is abortive, despite viral genome transcription and replication occurring for each gene segment and viral hemagglutinin and nucleoprotein, at least, being produced. Electron microscopy reveals that virus assembly, rather than release of virus from the cell surface, is defective.

Rapid generation of pandemic influenza virus vaccine candidate strains using synthetic DNA.

BACKGROUND: Vaccination is considered the most effective means of reducing influenza burden. The emergence of H5N1 and pandemic spread of novel H1N1/2009 viruses reinforces the need to have strategies in place to rapidly develop seed viruses for vaccine manufacture. METHODS: Candidate pandemic vaccine strains consisting of the circulating strain haemagglutinin (HA) and neuraminidase (NA) in an A/PR/8/34 backbone were generated using alternative synthetic DNA approaches, including site-directed mutagenesis of DNA encoding related virus strains, and rapid generation of virus using synthetic DNA cloned into plasmid vectors. RESULTS: Firstly, synthetic A/Bar Headed Goose/Qinghai/1A/2005 (H5N1) virus was generated from an A/Vietnam/1194/2004 template using site-directed mutagenesis. Secondly, A/Whooper Swan/Mongolia/244/2005 (H5N1) and A/California/04/09 (H1N1) viruses were generated using synthetic DNA encoding the viral HA and NA genes. Replication and antigenicity of the synthetic viruses were comparable to that of the corresponding non-synthetic viruses. CONCLUSIONS: In the event of an influenza pandemic, the use of these approaches may significantly reduce the time required to generate and distribute the vaccine seed virus and vaccine manufacture. These approaches also offer the advantage of not needing to handle wild-type virus, potentially diminishing biocontainment requirements.

Viral phenotypes and antibody responses in long-term survivors infected with attenuated human immunodeficiency virus type 1 containing deletions in the nef and long terminal repeat regions.

The Sydney Blood Bank Cohort (SBBC) consists of eight blood transfusion recipients infected with nef-attenuated human immunodeficiency virus type 1 (HIV-1) acquired from a single donor. Here, we show that viral phenotypes and antibody responses differ considerably between individual cohort members, despite the single source of infection. Replication of isolated virus varied from barely detectable to similar to that of the wild-type virus, and virus isolated from five SBBC members showed coreceptor usage signatures unique to each individual. Higher viral loads and stronger neutralizing antibody responses were associated with better-replicating viral strains, and detectable viral replication was essential for the development of strong and sustained humoral immune responses. Despite the presence of strong neutralizing antibodies in a number of SBBC members, disease progression was not prevented, and each cohort member studied displayed a unique outcome of infection with nef-attenuated HIV-1.

Broad neutralization and complement-mediated lysis of HIV-1 by PEHRG214, a novel caprine anti-HIV-1 polyclonal antibody.

OBJECTIVES: To assess the potency, breadth of action, and mechanism of action of the polyclonal goat anti-HIV antibody, PEHRG214. DESIGN: Typical human antibody responses to HIV-1 infection are unable to neutralize virus efficiently, clear the infection, or prevent disease progression. However, more potent neutralizing antibodies may be capable of playing a pivotal role in controlling HIV replication in vivo. PEHRG214 is a polyclonal caprine antibody raised against purified HIV-associated proteins, such that epitopes that are immunologically silent in humans may potentially be recognized in another species. It has been administered safely to HIV-infected individuals in Phase I clinical trials. METHODS: The anti-HIV activity of PEHRG214 was assessed using neutralization and virion lysis assays. The target proteins for PEHRG214 activity were investigated using flow cytometry and by adsorption of anti-cell antibodies from the antibody cocktail. RESULTS: PEHRG214 strongly neutralized a diverse range of primary HIV-1 isolates, encompassing subtypes A to E and both CCR5 and CXCR4 phenotypes. Neutralization was enhanced by the presence of complement. PEHRG214 also induced complement-mediated lysis of all HIV-1 isolates tested, and recognized or cross-reacted with a number of host cell proteins. Lysis was abrogated by adsorption with T and/or B cells expressing GPI-linked proteins, but not by GPI-deficient B cells or red blood cells. CONCLUSIONS: PEHRG214 was found to potently neutralize and lyse HIV-1 particles. By targeting host cell proteins present in the viral envelope, which are conserved among all strains tested, PEHRG214 potentially opens up a highly novel means of eliminating circulating virus in infected individuals.