Monash research targets vaccine against 'stealth microbe'

Researchers at Monash University have begun sifting through the database from Australia's first bacterial genome project, looking for targets for a vaccine against an unusual microbe that can kill, or leave survivors chronically depressed.

In Australia, the animal-transmitted agent of leptospirosis, the spirochaetal bacterium Leptospira, typically causes influenza-like symptoms and high fever. Traditional antibiotic therapy can be used to treat leptospirosis, but is of limited efficacy unless a diagnosis is made early during infection and treatment begins immediately.

Proteomicist Dr Paul Cullen, a member of Prof Ben Adler's leptospirosis research team at Monash University's Bacterial Pathogenesis Research Group, says most mammals -- including marine mammals -- have co-evolved with species-specific strains, or serovars, of Leptospira. The hosts can remain symptomless, but still transmit these strains to humans, causing severe illness. Leptospira has even been found in frogs.

Workers who have contact with animals are at highest risk -- leptospirosis is most common among dairy and pig farmers and veterinarians in temperate Australia.

In the sub-tropics, banana plantation workers are exposed to a more virulent form of leptospirosis through contact with bunches contaminated by rat urine. Most native rodents and marsupials tested to date have been found carry Leptospira. In Asia and South America, virulent, haemorrhagic Leptospira serovars kill thousands of people annually.

The corkscrew-shaped bacterium infects major organs, including the lungs. Causes capillaries to leak fluid into the tissues. Blood pressure plummets, and victims may die rapidly from multiple organ failure, or literally drown in their own body fluids.

Cullen says around 100,000 cases are reported annually, but leptospirosis is under-diagnosed, so the actual figure is at least three times higher.

Some 230 Leptospira serovars have been identified, but Cullen says the number probably runs to at least 400. The microbe's extreme antigenic diversity has frustrated efforts to develop a vaccine, so one of the main aims of the university's genome project is identifying strongly immunogenic antigens that may be conserved across many or all strains.

Using the latest 2D electrophoretic techniques and mass spectrometry, Cullen has been cataloguing and characterising all the microbe's outer membrane proteins.

"We're trying to detect differences in protein expression," he said. "Our first port of call will be proteins that are localized to the cell surface and that are expressed inside the host during infection.

"Alternatively, we might find that surface proteins that are only expressed outside the human host and that are vulnerable to antibody during the initiation of infection may be suitable vaccine candidates."

Promising candidates

The tick-transmitted spirochaete Borrelia burgdorferi, which causes Lyme disease, is the model for this approach to vaccine development, says Cullen.

"The first recombinant vaccine for Lyme disease employed a surface antigen that is not expressed in the human host -- only when Borellia is inside the tick," Cullen said. "When the tick ingests a blood meal from the human host, the antibodies in the blood immobilise the microbe in the tick's gut.

"It was a fortuitous discovery, because there is no selective pressure in the human host for the antigen to change."

There is no arthropod vector for Leptospira, but outer membrane antigens expressed at the beginning of the infectious process may still be promising candidates for a vaccine, says Cullen.

He describes Leptospira as a 'stealth' microbe -- some of its outer membrane proteins are inserted 'head down' in the membrane to evade the immune system, so are unpromising candidates for a vaccine.

The Monash team has been expressing the genes for some of these surface proteins in a specially designed E. coli lipidation vector. "When the genes are expressed from our vector, lipids are attached to the proteins, making them more immunogenic," says Cullen.

They have identified a promising antigen that they have dubbed 'mini-MOMP' (major outer membrane antigen). It's the second most abundant protein in the outer membrane, it's expressed both outside the host and during infection, and appears to be strongly immunogenic.

Testing prototype vaccines is tricky -- conventional rodent models are unsuitable, because the rat Leptospira serovars that infect humans are harmless to their rodent hosts, making it impossible to confirm that the vaccine is protective.

The best small animal model is the hamster -- but Cullen says hamsters can only be imported into Australia if they are desexed, making them extremely expensive. And the animals' immune systems are often too old by the time they arrive to determine whether the vaccine elicits immunity.

Hamster test

So the Monash team has been sending its vaccine-candidate antigens to Dr Richard Zuerner at the National Animal Disease Centre in Ames, Iowa, for testing in hamsters.

Zuerner's group has been doing its own Leptospira genome project on an isolate that is of the same serovar as the Monash groups strain but is pathogenic for different animal hosts. By comparing the genome data from the different isolates, the Monash and NADC teams hope to identify specific genes involved in virulence of different animal hosts.

Cullen's colleague, Dieter Bulach, has been assembling the genome data using nothing more than an off-the-shelf PC with a 1-gigahertz processor and extra RAM.

In 2001, the Monash team won a special project grant from the National Health and Medical Research Council, as part of a Commonwealth strategy to boost genomics research in Australia.

Bulach says he finished assembling the sequence last week, and the data will be published within the next few months.

Leptospira has two chromosomes -- a 3.6-megabase main chromosome with about 3200 genes, and a smaller, 320-kilobase chromosome with some 300 genes. The smaller chromosome resembles a large plasmid, but carries important 'housekeeping' genes that would normally reside on the larger chromosome.

Over the next few years the Monash group will continue to search for virulence genes and potential vaccine candidates by scanning their genome and proteome data sets.

Cullen says "We hope to receive further funding so that we can apply new technologies such as phenomics to characterise the numerous genes and their corresponding proteins, for which there appears no obvious function."