and immunological safety of DNA vaccines
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)
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
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 hosts 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.
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 Unions 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.
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-.
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
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.
September 1996 - October 1999
National Institute for Biological Standards and Control
Potters Bar (UK)
National University of Ireland
University of Bergen (NO)
Centro Nacional de Biología Fundamental
Institute for Animal Health