Pseudotype viruses - applications and troubleshooting - 2nd October 2013

Pseudotype viruses - applications and troubleshooting

Pseudotype viruses are rapidly establishing themselves as important research and diagnostic tools of basic and clinical scientists facilitating the detailed study of individual viral genes, host cell receptors and highly pathogenic viruses, circumventing the need for high-level biosafety containment. The switching of surface envelope proteins expressed on the surface of these pseudotypes enables them to be used as surrogate viruses in neutralization/antiviral screening assays and for the study of cell–virus receptor interactions. This meeting encompasses the many diverse applications of pseudotype technologies from a practical, translational and public health perspective. This event has CPD accreditation.

Meeting Chair: Dr Nigel Temperton, Senior Lecturer, University of Kent, UK

The Deadline for abstract submissions for oral presentation has now passed. Abstracts for poster presentation only can be submitted up to two weeks before the event. There will be a best poster prize.
You can download the instructions for authors at
www.euroscicon.com/AbstractsForOralAndPosterPresentation.pdf

Who Should Attend

• Public and animal health scientists.

• Individuals establishing diagnostic assays for viruses.

• Epidemiologists, virologists, immunologists, R&D, pre-clinical vaccinologists.

9:00 – 9:45 Registration

9:45 – 10:00 Introduction by the Chairs: Dr Nigel Temperton, Medway School of Pharmacy, The Universities of Greenwich and Kent, UK

10:00 – 10:30 Current progress with serological assays for exotic emerging/re-emerging viruses

Dr Janet Daly, Nottingham University, UK

Challenges exist in the development of serological assays for (re-)emerging viruses. Work with live virus is often restricted to specialised containment laboratories, thus limiting capacity to perform traditional serological assays such as the plaque reduction neutralisation test (PRNT). Diagnosis by ELISA-based assays using killed virus or purified or recombinant viral proteins offer an alternative. However, ELISA-based assays are often less specific than PRNT. Sample volume may be limited, for example where cerebrospinal fluid samples are required to confirm a viral cause of encephalitis. Pseudotype virus neutralisation assays offer the potential to address many of these issues.

10:30 – 11:00 Virus pseudotypes and pandemic preparedness

Dr Nigel Temperton, Senior Lecturer, University of Kent, UK

The availability of in-vitro cell culture based assays that can be readily employed for the efficacy testing of vaccines, antivirals and therapeutic antibodies are key components for effective pandemic preparedness. The exploitation of retroviral vectors pseudotyped with foreign heterologous envelope glycoproteins for the development of such assays will be discussed with particular empahasis on emerging influenza viruses.

11:00 – 11:30 Speakers’ photo then mid-morning break and trade show

11:30 – 12:00 Retroviral pseudotypes for equine influenza virus serology

Dr Simon Scott, Lecturer in Molecular Biology, University of Kent, UK

Standard assays for influenza serology present certain practical issues; interlaboratory variability, complex protocols and sometimes requirement to work in high biological containment. To address this, retroviruses pseudotyped with the haemagglutinin (HA) glycoprotein have been successfully employed in antibody neutralization assays. Recently, we generated the first equine influenza HA pseudotypes. This necessitated co-transfection with a specific endoprotease plasmid to cleave HA to produce infectious particles. PVs were then used in antibody neutralization assays to distinguish between vaccinated and non-vaccinated equines. Sera were screened in parallel by a standard single radial haemolysis assay. A 65% correlation was demonstrated between the two assays.

12:00 – 12:30 Interrogating the antibody response to rabies and lyssaviruses using retroviral pseudotyping

Dr Edward Wright, Westminster University, UK

Rabies virus is responsible for ~70,000 human deaths a year even though highly efficacious vaccines are available. A reliable figure for the number of deaths due to related lyssavirus species is unknown. Within the lyssavirus genus there are 11 species that can be classified into phylogroups, based primarily on their antibody cross neutralisation profile. As the lyssavirus glycoprotein is the major target of a neutralising antibody response a pseudotype-based neutralisation assay was developed to elucidate the importance of known glycoprotein epitopes in virus neutralisation. The assay has also proved useful for sero-epidemiological studies and testing existing and novel antivirals.

12:30 – 13:30 Lunch and trade show

13:30 – 14:00 Dissecting serological responses to ruminant viruses using retrovirus pseudotypes

Dr David Griffiths, Principal Research Scientist, Moredun Research Institute, Pentlands Science Park, UK

Retroviral pseudotypes are valuable tools for investigating serological responses to viruses, particularly in cases where a permissive culture system is not available. An example is jaagsiekte sheep retrovirus (JSRV), which causes ovine pulmonary adenocarcinoma, a fatal lung cancer of sheep. Until recently, it was thought that infected sheep cannot produce an adaptive immune response to JSRV and this has hindered the generation of vaccines and diagnostics for controlling the disease. Using pseudotypes, we have shown that some infected sheep elicit neutralising antibodies to JSRV, opening up new opportunities for the development of control strategies for this important veterinary pathogen.

14:00 – 14:30 Quantifying the infectivity and neutralisation of companion animal viruses using retroviral pseudotypes

Professor Brian Willett, University of Glasgow Scotland, UK

The study of viral diseases of many species is hindered by the paucity of reagents with which viral infectivity may be measured. The ability to generate retroviral pseudotypes bearing envelope glycoproteins from diverse viral genera offers a unique opportunity to investigate hitherto intractable questions in viral pathogenesis. Pseudotypes bearing FIV and FeLV Envs have been used to elucidate viral receptors, to quantify neutralising antibodies and to stage clinical disease, while pseudotypes bearing lyssaviral glycoproteins have facilitated the detection of neutralising antibodies in sera from wild carnivores. Retroviral pseudotypes offer a novel means to broaden our understanding of viral pathogenesis.

14:30 – 15:00 Afternoon Tea/Coffee and trade show

15:00– 16:00 Question and Answer Session

16:00 - 16:30 Use of HIV Pseudovirions to investigate susceptibility to Antiretrovirals

Katherine Sutherland, Microbiology Services, Colindale Public Health England, UK

Research using HIV pseudovirions can be carried out at Containment Level 2 making research quicker, safer and cheaper. We use pseudovirions based on the HIV Gag-Pol expression vector, p8.9NSX, which encodes the HIV structural proteins and enzymes necessary for viral replication. Patient-derived HIV genes can be inserted into p8.9NSX+ separately or in combination. Pseudovirions comprise a non-HIV envelope glycoprotein enabling infection of common cell types and an HIV-based genome encoding the luciferase reporter gene to enable quantification of infection. We use pseudovirions to investigate susceptibility to different antiretrovirals, determine viral replicative fitness as well as viral structure using electron microscopy.

16:30 - 17:00 Retroviral pseudotypes for investigating the humoral immune response to diverse hepatitis C virus strains

Dr Alexander Tarr, Senior Research Fellow, School of Life Sciences, University of Nottingham, UK

Hepatitis C virus (HCV) causes a chronic infection that can result in cirrhosis and liver cancer. Entry of HCV into host cells is mediated by virus glycoproteins E1 and E2. Although entry is an attractive target for therapeutic intervention and vaccine design, the extreme diversity (>30% difference in nucleotide sequence between isolates) exhibited by these proteins presents a significant challenge. We have investigated entry of retroviral pseudotypes possessing genetically diverse HCV glycoproteins to identify broadly neutralizing anti-E1/E2 antibodies that target conserved epitopes. This strategy has revealed key entry steps that can be targeted for therapeutic intervention.

17:00 Chair’s Summary and Close of Meeting

Registration Website: www.regonline.co.uk/pseuVirus2013

About the Chair

Nigel Temperton obtained his BSc in Microbiology and Genetics from UCL in 1990 and an MSc in Applied Molecular Biology of Infectious Diseases (1992), PhD in Molecular Parasitology (1999) and DLSHTM (2000) from LSHTM. After his PhD, Nigel returned to UCL as a post-doctoral scientist at the Centre for Virology. In 2003 Nigel transferred to the MRC/UCL Centre for Medical Molecular Virology initially as a senior post-doctoral scientist and subsequently Principal Investigator, funded by the MRC and industry. He is currently a Senior Lecturer at the Medway School of Pharmacy and Principal Scientist at the Viral Pseudotype Unit.

About the Speakers

Janet Daly gained a PhD for studies on the antigenic and genetic variation of equine influenza viruses at the Animal Health Trust (AHT) in Newmarket. She subsequently worked on human influenza projects at the National Institute for Biological Standards and Control and GlaxoSmithKline. She returned to the AHT to lead the equine influenza surveillance and control programme there for several years before moving to the University of Liverpool to work with Japanese encephalitis virus. She joined the University of Nottingham’s School of Veterinary Medicine and Science in 2009 where her main research interests are zoonotic RNA viruses.

Simon Scott began his research career as a DNA virologist, working in Cambridge and Newmarket on the molecular biology of animal herpesviruses. Following an EU fellowship in Amsterdam studying human papillomavirus oncology, he spent over a decade undertaking research in the field of cancer gene therapy using DNA and RNA virus delivery vectors, in both the UK and USA. After joining the University of Kent he established the Viral Pseudotype Unit with Dr Nigel Temperton. The focus of his pseudotype worksince has been on neglected influenza viruses and more recently emerging RNA viruses from other virus families (e.g. flaviviruses, bunyaviruses).

Edward Wright is a Principal Scientist at the Viral Pseudotype Unit labs in central London. After completing his BSc in Virology at the University of Edinburgh, Edward successfully studied for a PhD in Molecular Virology from the University of Cambridge. He subsequently obtained a position at the Medical Research Council/Uganda Virus Research Institute Research Unit on AIDS. This was followed by a period as Research Fellow at University College London where he furthered the understanding of highly pathogenic viruses such as rabies/lyssaviruses and HIV, primarily using pseudotypes as the tools for these studies. As a Lecturer in Medical Microbiology at the University of Westminster, Edward’s research continues into the pathogenicity and antigenicity of viral zoonoses.

David Griffiths graduated from the University of Manchester in 1992 with a B.Sc. in Biochemistry. He gained a Ph.D. in Virology from the University of London in 1996 for his studies on the potential role of retroviruses in human rheumatic disease. Following a post-doctoral position at the Chester Beatty Laboratories London, in 1998 he was awarded an Arthritis Research Council Postdoctoral Fellowship at University College London. In 2003, Dr Griffiths moved to the Moredun Research Institute as a Principal Research Scientist to study the pathogenesis of ovine pulmonary adenocarcinoma and the development of control methods for this disease.

Brian Willett graduated from the University of Strathclyde with a BSc (Hons) in Biochemistry and Pharmacology, and a PhD in Immunology. He joined the University of Glasgow in 1989, working with Prof. Oswald Jarrett on the immunopathology of feline leukaemia virus infection. Subsequent studies on the interaction between feline immunodeficiency virus and the cat immune system led to the characterization of the virus-receptor interaction. Current research interests include intrinsic and adaptive immunity to felid retroviruses, viral vaccine development, and novel approaches to the measurement of humoral immunity in wild felids and other animals.

Katherine Sutherland is currently studying for a PhD at Public Health England in Colindale, London and University College London. Her PhD has involved the investigation of susceptibility of HIV viruses to protease inhibitors using pseudovirus based assays. Her work has been presented at national and international conferences.

Alexander Tarr graduated with a PhD in molecular virology from the University of Nottingham. His postdoctoral research focussed on the entry pathway of hepatitis C virus (HCV) and characterizing the specificity of the antibody response during chronic HCV infections. This collaborative research revealed common entry pathways for HCV, and subsequently identified monoclonal antibodies that potently neutralize entry of genetically diverse HCV strains. His current research examines the interplay between innate and adaptive immunity in chronic viral infections, especially the contribution of soluble pattern recognition receptors to virus neutralization.

Post expires at 8:03am on Wednesday October 2nd, 2013

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