Mosquito-borne virus helps in vaccine vector quest

By Graeme O'Neill
Monday, 04 April, 2005

A new Brisbane biotech, RepliKun, has been spun out from the Queensland Institute of Medical Research (QIMR), the University of Queensland and the state's health department to commercialise a novel vaccine vector, based on the Kunjin virus replicon -- the self-replicating genetic core of the mosquito-borne virus of Australia's tropics.

QIMR team leader Dr Andreas Suhrbier said the Kunjin replicon is an RNA based gene-delivery system with unique features that suit it for developing vaccines against cancer or HIV/AIDS, and for gene therapy.

One of the genes in the Kunjin replicon codes for a replicase enzyme that creates a negative-strand RNA template for the infected cell to make new positive-strand copies of the replicon, along with any transgene spliced into it.

Suhrbier said experiments have shown that the replicon can accommodate a large range of gene sizes. Researchers have tested about 20 genes so far, the largest being the beta galactosidase gene, which encodes an enzyme widely used to visualise gene expression patterns in plant and animal tissues.

Shorn of the genes coding for the Kunjin virus coat proteins, the single-stranded RNA replicon is permanently contained within the cytoplasm of transfected cells -- it does not integrate into the nucleus, and cannot infect healthy cells. However, every time the transfected cell divides, the replicon, plus any transgenes, is installed in its progeny.

Suhrbier said the biggest advantage of the Kunjin replicon is that, unlike the rival alpha virus replicon, its rapid replication does not induce programmed cell death, or apoptosis.

When the transfected cells die, synthesis of transgenic proteins ceases. The Kunjin replicon produces proteins that suppress the interferon response, which Suhrbier suspects is the reason the cells do not die.

In contrast to the alpha virus replicon, which persists for only a few weeks, the Kunjin replicon appears to persist for life in a mouse model.

By establishing a positive feedback loop, the replicase enzyme produces more and more copies of itself, each coding for more replicase. The incorporated transgene is also over-expressed, and the cells continuously produce high concentrations of vaccine antigens -- a very desirable property for vaccines and gene therapy, said Suhrbier.

The replicon shows no tissue specificity -- it transfects a diverse panel of cancerous cell lines. It also elicits a strong cytotoxic T-cell response to the encoded transgenic antigen -- exactly the type of response required for the immune system to efficiently detect and destroy cancerous cells.

To make a therapeutic vaccine for cancer, a transgene coding for some protein -- normal or mutant -- specific to that cancer is spliced into the replicon, and used to transfect memory T-cells. The antigen is processed by dendritic cells, which present the antigen to cytotoxic T-cells, priming them to seek out and destroy any cancerous cells displaying the antigen.

Suhrbier said such vaccines would be useful to mop up residual cancerous cells after surgery or other cancer therapies, to prevent them developing into secondary cancers. The QIMR research team has been using ovalbumin as a proxy for a cancer antigen, and has achieved an "extraordinary" cytotoxic T-cell response, Suhrbier said.

RepliKun also plans to develop the system to produce bulk quantities of diagnostics and therapeutic proteins in mammalian cell-culture systems.

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