Accelerating Influenza Research: Vaccines, Antivirals, Immunomodulators and Monoclonal Antibodies. The Manufacture of a New Wild-Type H3N2 Virus for the Human Viral Challenge Model

Abstract

Background: Influenza and its associated diseases are a major cause of morbidity and mortality. The United States Advisory Committee on Immunization Practices recommends influenza vaccination for everyone over 6 months of age. The failure of the flu vaccine in 2014-2015 demonstrates the need for a model that allows the rapid development of novel antivirals, universal/intra-seasonal vaccines, immunomodulators, monoclonal antibodies and other novel treatments. To this end we manufactured a new H3N2 influenza virus in compliance with Good Manufacturing Practice for use in the Human Viral Challenge Model.

Methods and strain selection: We chose an H3N2 influenza subtype, rather than H1N1, given that this strain has the most substantial impact in terms of morbidity or mortality annually as described by the Centre for Disease Control. We first subjected the virus batch to rigorous adventitious agent testing, confirmed the virus to be wild-type by Sanger sequencing and determined the virus titres appropriate for human use via the established ferret model. We built on our previous experience with other H3N2 and H1N1 viruses to develop this unique model.

Human challenge and conclusions: We conducted an initial safety and characterisation study in healthy adult volunteers, utilising our unique clinical quarantine facility in London, UK. In this study we demonstrated this new influenza (H3N2) challenge virus to be both safe and pathogenic with an appropriate level of disease in volunteers. Furthermore, by inoculating volunteers with a range of different inoculum titres, we established the minimum infectious titre required to achieve reproducible disease whilst ensuring a sensitive model that can be translated to design of subsequent field based studies.

Trial registration: ClinicalTrials.gov NCT02525055.

Fig 1. Summary of Clinical Trial Design.

Fig 2. Summary of Production of the GMP Challenge Stock.

Fig 3. Virus shedding (qPCR) by inoculum titre group in the Ferret Viral Challenge Model.

Fig 4. Histopathology of the Ferret Lung Post Challenge with A/Perth/16/2009 or A/Wisconsin/67/2005.

A) Ferret Lung Day 4 post inoculation with A/Wisconsin/67/2005 2.5. × 10⁴ TCID₅₀/animal. B) Ferret Lung Day 7 post inoculation with A/Wisconsin/67/2005 2.5 × 10⁴ TCID₅₀/animal. C) Ferret Lung Day 4 post inoculation with A/Perth/16/2009 1.8 × 10⁵ TCID₅₀/animal. D) Ferret Lung Day 7 post inoculation A/Perth/16/2009 1.8 × 10⁵ TCID₅₀/animal. Alveolar lumina were filled with hyaline eosinophilic proteinaceous material (edema, see arrows).

Fig 5. Mean Viral Shedding in qPCR positive subjects in the Human Viral Challenge Model.

Fig 6. Mean viral shedding by day in TCID₅₀ positive subjects in the Human Viral Challenge model.

Fig 7. The impact of inoculum titre on viral load in infected subjects.

Fig 8. Progression of symptoms over the course of infection in all subjects, by titre group (infected and non-infected volunteers).

Fig 9. Mean symptom score by day in symptom positive subjects in the Human Challenge Viral Model.

Fig 10. The impact of inoculum titre on symptomology in subjects positive for symptoms.

Fig 11. Mean mucus weight by day in all subjects in the Human Viral Challenge Model.

Fig 12. The effects of virus inoculum titre on virus shedding and symptoms in the chosen inoculum titre group in all subjects (infected and non-infected volunteers).