Archive for June 2nd, 2008

West Nile virus vaccine: almost a replicant

2 June, 2008

West Nile virus – a member of the family Flaviviridae – has insidiously spread halfway around the world from its origins in Africa, in just a few years.  It invaded the east coast of the USA, probably from the Middle East,  via either infected birds, mosquitoes, humans, or another vertebrate host in around 1999; since then it has spread all the way across the continent to the west coast, and has become truly endemic. 

Virions have a regular icosahedral-type structure, despite being enveloped, as a result of a structured nucleocapsid and a highly-structured array of envelope glycoprotein.  They contain a positive strand RNA genome of ~11 kb with a single long open reading frame that is translated as a polyprotein of about 3400 amino acids, which is then processed into individual regulatory and structural proteins.

The virus subtype spreading in North America – lineage 1 – causes encephalitis in humans, unlike the enzootic variant circulating in birds and animals in Africa.  It also cause severe mortality – near 100% in experimentally infected animals – among American Crows and other corvids: a feature of the spread of the disease has been dead crows found in and around towns in the USA.  A feature of lineage 1 viruses is their infection of horses and other equines as well – with up to one in three clinically-infected horses dying.  The human impact, however, is seen as a major problem: systemic febrile illness develops in ~20% of those infected with WNV, while severe neurologic illness developes in <1% of persons infected – with mortality rates of 5 -14% among persons with neurologic symptoms in recent US, Romanian, Russian, and Israeli outbreaks.

There has been a concentrated effort to develop a human vaccine or vaccines since the onset of the US epidemic – horse vaccines are already commercially available – and our knowledge of the virus has benefitted greatly as a result.  This includes a detailed structure for the virus, obtained by cryoelectron microscopy image reconstruction.

Purdue team solves structure of West Nile virus via kwout

 Now a team led by Alexander Khromykh from Brisbane in Queensland, Australia, writing in the May issue of Nature Biotechnology, have described a novel “single-round infectious particle” DNA vaccine against WNV which significantly increases protection in mice to lethal challenge with the live virus.   In the words of the authors:

“We augment the protective capacity of a capsid-deleted flavivirus DNA vaccine by co-expressing the capsid protein from a separate promoter. In transfected cells, the capsid-deleted RNA transcript is replicated and translated to produce secreted virus-like particles lacking the nucleocapsid. This RNA is also packaged with the help of co-expressed capsid protein to form secreted single-round infectious particles (SRIPs) that deliver the RNA into neighboring cells. In SRIP-infected cells, the RNA is replicated again and produces additional virus-like particles, but in the absence of capsid RNA no SRIPs are formed and no further spread occurs. Compared with an otherwise identical construct that does not encode capsid, our vaccine offers better protection to mice after lethal West Nile virus infection. It also elicits virus-neutralizing antibodies in horses. This approach may enable vaccination against pathogenic flaviviruses other than West Nile virus.”

Adapted by permission from Macmillan Publishers Ltd: Nature Biotechnology 26, 571 – 577, 20 April 2008 doi:10.1038/nbt1400 Single-round infectious particles enhance immunogenicity of a DNA vaccine against West Nile virus, David C Chang et al., copyright 2008

This is a very clever use of fundamental knowledge of virus structure and assembly: the virus envelope proteins – E and prM – can form budded particles if expressed in isolation; if expressed with the capsid protein, the particles encapsidate RNA with the appropriate encapsidation signal to form virions.  The DNA vaccine encodes a transcriptional unit corresponding to a viral genome which lacks only the capsid protein gene, as well as a separate capsid gene under back-to-back cytomegalovirus (CMV) promoters.  Thus, cells transfected with the DNA vaccine can produce both virus-like prM and E protein and membrane particles (VLPs), or pseudovirions which in addition contain a capsid and the engineered (=lacking capsid protein gene) genome.  While both are highly immunogenic, the pseudovirions can additionally infect other cells to release replicative genomic RNA, which can produce VLPs but not pseudovirions, as the capsid protein-encoding RNA is not encapsidated.  Thus, initial transfection leads to release of particles which allow a single subsequent round of VLP production, but no further spread of the replicative RNA.

A very clever trick – and worthy of being repeated for a number of related pathogenic flaviruses, including dengue and yellow fever viruses.

Even if the particles can’t pass the Voight-Kampff test…B-)