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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Genetic and immunological safety of DNA vaccines

Background and objectives

DNA plasmid injection is a novel approach to vaccination which has received considerable attention in recent years. However, immunisation with plasmid DNA raises novel safety issues. Potential immunological problems associated with this form of vaccination include unexpected immunopathological reactions or tolerance of the vaccine. Researchers are also investigating the co-administration of regulatory hormones to improve immune responses. However, this may have even more serious consequences, with the possibility of stimulating one arm of the immune response at the expense of the other, leading to generalised immunosuppression or chronic inflammation. Another serious, although somewhat hypothetical, concern is that the injected DNA may integrate into a host chromosome resulting in a transformation (or tumourigenic) event.

The objectives of this project were to investigate circumstances which may lead to an undesirable immune response to a DNA vaccine and to establish an assay for assessing the integration of a plasmid DNA vaccine.


Approach and methodology

DNA vaccines composed of genes encoding antigens from influenza virus and from respiratory syncytial virus (RSV) under the control of a eukaryotic promoter were used. An efficient DNA plasmid expression vector, designated pI.17, was constructed for this study, along with newer variants, pI.18 and pI.19. These were engineered to contain different viral genes, and also different cytokines. In a comparative study alongside commercially available expression vectors, vector pI.17 showed the highest expression in vitro, and the greatest antibody response in vivo in mice.


Main findings and outcome

Using the influenza model, it was clear that intramuscular (i.m.) DNA vaccination produced a potent Th1-biased immune response, which was only long lasting when large quantities and multiple doses of DNA were used. This response appears to involve activation of splenic T cells by antigen presenting cells which were present in the spleen as soon as one hour after the inoculation. Adverse inflammatory responses were investigated by the co-administration of bacterial lipopolysaccharide (LPS). DNA virtually free of LPS, appeared to exert an inhibitory effect on the production of inflammatory cytokines, whereas low doses of co-administered LPS augmented inflammatory responses. Therefore, the degree of purity of plasmid DNA could result in widely differing effects.

RSV infection following immunisation, has previously been shown to cause immune-mediated lung pathology. Intramuscular DNA vaccination resulted in reduced lung pathology following RSV challenge, and protection against infection. Co-administration of DNA encoding cytokines could influence the type of immune response, without inducing any immuno-pathological reactions. Gene-gun inoculation was also highly effective in inducing an immune response, although the nature of the response was strongly biased towards a Th2 response, irrespective of the RSV antigen. Furthermore, gene-gun vaccination primed for a pulmonary eosinophil response after RSV challenge. Thus, the Th2 environment induced by gene-gun vaccination appears to influence the pulmonary inflammatory response to subsequent RSV infection. It is therefore important to further analyse the potential for adverse reactions following gene-gun vaccination.

There remains considerable concern that a DNA plasmid vaccine may integrate into a host’s chromosomes and initiate a tumourigenic event. It was important to establish and implement methods currently being used by other groups to assess chromosomal integration of a DNA vaccine. As other groups have reported, we did not detect integration in an experimental model. However, in contrast to other reports, plasmid DNA was found to persist at the site of inoculation one year later, although there was no evidence for expression from these remaining plasmids.


Conclusions

Our studies have shown that the purity of injected DNA vaccines can affect the inflammatory response, and also that the type of vaccination can influence the nature of this response. This data will benefit the European scientific community through its contribution to our knowledge of the underlying mechanisms of DNA vaccines and of a greater understanding of the potential for DNA vaccines to cause adverse immunopathological reactions. Furthermore, two members of this project are on the Biotech Working Party which advises the European Union’s Committee for Proprietary Medicinal Products on the safety and efficacy of biologicals, including vaccines. Two other partners provide expert advice to national control authorities. Without the "hands-on" experience which these partners now have, accurate, effective scientific advice cannot be provided. This is necessary not only to ensure proper regulation of DNA vaccines but also to limit the possibility of ill-informed opinions impeding the passage of a clinical trial submission or licence application, which would thus be to the detriment of the vaccine industry and consequently to the general public.

 

Major publications

Bembridge G.P., López J.A., Cook R., Melero J.A. and Taylor G., “Recombinant vaccinia virus co-expressing the F protein of respiratory syncytial virus (RSV) and IL-4 does not inhibit the development of RSV-specific memory CTL whereas priming is diminished in the presence of high levels of IL-2 and INF-epsy”.
J. Virol.,
72, 1998, p. 4080.

Bembridge G.P., García-Beato R., López J.A., Melero J.A. and Taylor G., “Subcellular site of expression and route of vaccination influence pulmonary eosinophilia following respiratory syncytial virus challenge in BALB/c mice sensitized to the attachment protein”.
J. Immunol.,
161, 1998, p. 2473.

Nicolson C., Lloyd P., Marsden S., Minor P.D. and Robertson J.S., “Is Plasmid DNA Tumourigenic?”, in Proceedings of 2nd EC Biotechnology Meeting on Vaccinology, P. Roy & C. Leclerc (eds.), 1998, pp. 73-74.

Johnson P.A., Conway M.A., Daly J., Nicolson C., Robertson J. and Mills K.H.G., “Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses”.
J. Gen. Virol., 81, 2000, pp. 1737-1745.

Bembridge G.P., Rodriguez N., Garcia-Beato R., Nicolson C., Melero J.A. and Taylor G., “DNA encoding the attachment (G) or fusion (F) protein of respiratory syncytial virus induces protection in the absence of pulmonary inflammation”.
J. Gen. Virol., 81, 2000, pp. 2519-2523.

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imageResearch project
 

Contract number
BIO4-CT96-0637

Period
September 1996 - October 1999

Coordinator
J.S. Robertson
National Institute for Biological Standards and Control
Potters Bar (UK)

 
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Partners


K.H.G. Mills
National University of Ireland
Maynooth (IE)

L. Haaheim
University of Bergen (NO)

J.A. Melero
Centro Nacional de Biología Fundamental
Majadahonda (ES)

G. Taylor
Institute for Animal Health
Compton (UK)

 
 
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