4th Year MPharm SRP: Introduction to Pseudotype Viruses

Stuart Mather

Project Leaders: Dr Nigel Temperton and Dr Simon Scott

Influenza virus

• Seasonal influenza - WHO estimate = 1 billion cases, 3-5 million severe illnesses, 300-500k deaths

• Huge financial implications – treatment and vaccination costs, loss in workforce productivity

• Certain subtypes responsible for pandemic influenza outbreaks – e.g. Spanish flu (H1N1) – death toll of approx. 50 million in 10 months

• Member of the Orthomyxoviridae family of viruses

• 3 genera = Influenza A, B & C

http://www.nhs.uk/Conditions/vaccinations/Pages/flu-influenza-vaccine.aspx

National Museum of Health and Medicine, Armed Forces Institute of Pathology, Washington, DC, USA (NCP1603).

Influenza virion• RNA genome – eight segments, single stranded, negative sense

HA = responsible for binding of virus to sialic acid cellular receptor

NA = cleaves sialic acid to release progeny virus during egress

17 subtypes of HA and 10 subtypes of NA:

http://en.wikipedia.org/wiki/Influenza

Taken from ‘Fields Virology’ (Eds Knipe & Howley)

Influenza A Virus Transmission

Antigenic drift• Accumulation of random mutations in epitopes of

viral antigens

• ‘Evolution’ of HA so that existing antibodies less efficient at neutralising drift variants

http://www.rapidreferenceinfluenza.com

• Cause of recurrent influenza epidemics – necessary to repeatedly vaccinate

http://www.virology.ws

Antigenic shift• Introduction of new or evolved influenza subtype into the human population

– Direct avian to human transmission– Reintroduction of historic virus subtype– Genetic reassortment

• Cause of devastating global influenza pandemics - population has totally naïve immunity to the novel virus

Serological assays• Serology – study of plasma serum – identification of antibodies

• PRNT – ‘gold standard’ for measuring virus-neutralising antibody (VNAb) response– Required to use infectious virus– Low-throughput – takes ~6 days to produce assay results

• Other assays, such as HI, SRH and ELISA are employed– Do not measure neutralising antibody response – Issues with low sensitivity and specificity in some cases

• Need for a sensitive and specific method for measure VNAb response without handling live virus

• Pseudotype viruses are a solution!

Pseudotype viruses

• ‘Chimeric’ viruses made up of a retroviral core (e.g. HIV), a heterologous envelope (e.g. influenza HA) and encapsulating a quantifiable reporter gene (e.g. luciferase)

• HIV - core• Influenza HA – envelope• Luciferase – reporter

• Non-infectious - Can be used instead of infectious virus in serological assays to determine neutralising antibody titres

Cloning of HA gene - plasmids

http://www.addgene.org/plasmid_protocols/subcloning/

Pseudotype virus production

Mather et al (2013) Future Virology 8(8); 745-755

Reporter gene flexibility

• Variety of reporters can be incorporated into the pseudotype platform

• Ability to ‘cost-customise’ the system

Temperton and Wright (2009) Encyclopedia of Life Sciences