Reverse genetics to map determinants of Varicella zoster Oka vaccine attenuation and virulence.

Chickenpox is a common infection of childhood which is usually uncomplicated. Following infection, the causative virus, known as varicella-zoster virus (VZV) remains dormant within the nerves reactivating at a later date in some people to cause shingles. In adults and in people with depressed immunity (HIV+ve, transplant patients, underlying congenital immunodeficiency, or receiving immunosuppressive drugs) chickenpox is more commonly fatal. A vaccine, called vOka, is licensed for the prevention of chickenpox and shingles and is widely used in the USA, parts of Europe and Canada. Although extremely safe it causes a rash in about 5% of healthy vaccinees. Unfortunately the vaccine can also cause life threatening disease in those who are already most at risk of ordinary chickenpox, namely immunosuppressed patients. Since these are the patients would be likely to benefit most from immunisation, a safer vaccine is urgently required. Previous work has identified that the vOka vaccine contains mixture of closely related variant viruses. We have shown that only a few of the variants within the vaccine preparation are responsible for causing vaccine rashes. These rash-forming vaccine variants are more likely to carry certain mutations within one gene known as ORF62. At the same time, we have access to an experimental batch of the Oka vaccine which was never licensed because it caused too many rashes. Finally, we have identified a case in which the vaccine virus appeared to become virulent, transmitting from a child with a rash to his parent who developed a full blown case of chickenpox. We now propose to look at the genetic sequences of these rash-causing viruses and compare them with the vaccine itself and the original virus from which the vaccine was made. We will examine whether these more virulent vaccine viruses behave differently in models of skin and develop new methods to assess whether certain proteins within the virus including the protein coded for by ORF62 are altered in the vaccine and in the rash-causing vaccine viruses. Importantly, the results will provide information which will help in the development of new, improved vaccines to prevent chickenpox and shingles.

The aim of this proposal is to investigate, using whole genome sequencing, reverse genetics and in vitro assays of replication and function,the viral determinants of Oka varicella-zoster vaccine strain attenuation and virulence. The Oka vaccine is licensed in the USA and other countries for immunisation of children to prevent chickenpox. Although safe and effective, the vaccine can cause life-threatening infection if given to patients with impaired cell-mediated immunity. Since these patients are at greatest risk of complications and death from chicknpox, newer safer vaccines are still needed. There is no good animal model which adequately replicates VZV propagation and virulence in vivo. This has hampered attempts to link vaccine mutations with clinical phenotype. We have shown, using a statistical genetics approach, that despite attnuation of the Oka vaccine for replication in skin, some vaccine variants within the vaccine mix are rash-forming. In a separate study, we have identified an altered batch of pre-licensure vOka vaccine, which caused an unusually high number of rashes. Finally, we have obtained a vOka virus which transmitted from a child with vaccine-rash, to cause fully virulent chickenpox. These viruses provide a valuable resource with which to examine the genetic determinants of Oka vaccine attenuation and VZV pathogenicity. To exploit this unique clinical material, we propose developing PCR-based genome sequencing. This will make use of the small amounts of viral DNA available and will establish a proof of concept applicable to other viral genomes. We will use techniques established in our lab to reconstruct viruses of interest and to assay their replication in models of differentiated epithelia. The location of over 1/3 of vaccine mutations in the gene coding for the major viral transactivating protein IE62, together with the association of changes in IE62 with rash formation implies, an important role in viral virulence. Functional differences between IE62 molecules using currently available reporter assays are not well detected. We now propose to use time lapse confocal microscopy of fluorescent-tagged molecules to investigate IE62 distibution and recruitment of celluar proteins in early infection . Similar experiments using the Herpes simplex virus (HSV) homologue of IE62 suggest that altered proteins are distributed differently early in infection and may function less efficiently.
This project will allow mapping of the vaccine mutations associated with clinical virulence and the development of in vitro assays of viral function and replication. The results will inform studies to improve vaccine safety.