Going ballistic

"Conventional antivenom preparations are relatively crude preparations which produce many antibodies against non-toxic as well as toxic molecules in snake venom," explains Robert Harrison. "We hope to make something much more specific."

At present, antivenoms are produced using age-old techniques. Animals, usually horses, are injected with venom to stimulate an antibody response, and the antibodies are then extracted from the horse serum and purified. This method is not cheap, a factor that is contributing to a critical shortage of antivenoms in Africa. Neither are the remedies totally effective. Moreover, Dr Harrison points out, "large amounts have to be given to the patient and they can have serious side-effects."

In Liverpool, Dr Harrison and collaborators Professor David Theakston and Gavin Laing are attempting to produce cheaper and more effective antivenoms by targeting only the toxic components of venom and by using the relatively new technique of DNA immunisation. In this approach, DNA sequences representing fragments of the genes encoding key toxin molecules are injected and act as templates for the production of protein. The body’s own cells make the fragments of toxin protein, which are not themselves harmful but still stimulate an immune response.

The team carried out preliminary studies on the venom of the Brazilian pit viper (Bothrops jaracaca), which causes serious and sometimes lethal bleeding. Mice were immunised with part of the jararhagin gene, which encodes a powerful enzyme that both causes bleeding and prevents blood from clotting. The DNA was delivered using a ‘gene gun’, a needleless vaccine delivery system pioneered by PowderJect Pharmaceuticals in which microscopic gold beads coated with DNA are shot into the skin. This needle-free, pain-free delivery system successfully delivered the truncated jararhagin gene, and the immunised mice responded by producing potent anti-jararhagin antibodies. Most encouragingly, these antibodies were nearly as effective as sera made from whole venom in neutralising viper venom.

With further Trust support, Dr Harrison is extending the molecular approach to the carpet viper (Echis). Carpet viper venom also causes bleeding and disrupts clotting, and an Echis bite is frequently fatal. "Carpet vipers are responsible for more deaths than any other genus of snakes in the world," says Professor Theakston.

The aim is to immunise mice with DNA encoding only the pathogenic molecules in the Echis venom, in order to generate a panel of antibodies capable of neutralising all its toxic activity. The first step will be to identify the gene sequences encoding these active molecules, which include metalloproteineases that degrade the blood vessel wall, disintegrins that inhibit platelet aggregation, and other components that prevent blood coagulation. These DNA sequences will then be manipulated to ensure that their expression will pose no threat to the host but will stimulate potent antibody responses. The group will again use the gene gun to deliver DNA, but will also administer DNA encoding cytokines (proteins that boost immune responses).

If the resulting sera are able to neutralise venom, Dr Harrison plans to develop the technique further and produce ‘humanised’ antibodies – mouse antigen-binding sites fused to human antibodies – which would not be seen as ‘foreign’ in human recipients, thus avoiding the side-effects associated with horse antivenom. This would also allow the antibodies to be produced by cells grown in culture. "It will be an immortalised source," says Dr Harrison. "They’ll be frozen down, ready for whenever we need them. We’ll have them forever."

This new method of antivenom production by DNA immunisation should be at least as effective, if not better, than the conventional antivenoms. "They should be more toxin-specific with reduced risks of side-effects," says Dr Harrison. They should also be more cost-effective, which would be particularly beneficial in the developing world where antivenoms are most needed but least affordable .

See also

• Spot the difference: Article describing research on the taxonomy of pit vipers through DNA analysis
• Screen test: Article describing research into traditional Nigerian snakebite remedies

External links

• Robert Harrison: Venom and toxin research at the Liverpool School of Tropical Medicine
• BBC news article: DNA 'stems anti-venom shortage' 31 August 2000

Further reading

Harrison R A, Moura-Da-Silva A M, Laing G D, Wu Y, Richards A, Broadhead A, Bianco A E, Theakston R D (2000). Antibody from mice immunized with DNA encoding the carboxyl-disintegrin and cysteine-rich domain (JD9) of the haemorrhagic metalloprotease, Jararhagin, inhibits the main lethal component of viper venom. Clinical & Experimental Immunology 121(2): 358–63.

Harrison R A, Bianco A E (2000). DNA immunization with Onchocerca volvulus genes, Ov-tmy-1 and OvB20: serological and parasitological outcomes following intramuscular or GeneGun delivery in a mouse model of onchocerciasis. Parasite Immunology. 22(5): 249–57.

Harrison R A, Wu Y, Egerton G, Bianco A E (1999). DNA immunization with Onchocerca volvulus chitinase induces partial protection against challenge infection with L3 larvae in mice. Vaccine. 18(7–8): 647–55.

Abdeen H H, Attallah A F, Mansour M M, Harrison R A (1999). Molecular cloning and characterization of the polypeptide backbone of Schistosoma mansoni circulating cathodic antigen. Molecular & Biochemical Parasitology. 101(1–2): 149–59.

Abdeen H H, Attallah A F M, El-Mohamady H I, Harrison R A, Mansour M M (1998). Schistosoma mansoni: the circulating cathodic antigen forms an abundant product of 41/42 kDa in the urine of infected patients. Experimental Parasitology. 90(3): 286–9.