Nucleic acid vaccines

Parasitology Today,vol. IO, no. 12, I 994

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Nucleic

Acid Vaccines G.J. Waine

Geneva, Switzerland May 1994 One of the most exciting new developments in vaccinology is the advent of nucleic acid vaccines, which are attractive because of their relative simplicity. A gene encoding a protective antigen is cloned into a plasmid co~~struct containing a eukaryotic promotor. This construct is then delivered to the host to be vaccinated, usually by direct injection into the muscle. By still ill-defined mechanisms, the nucleic acid is taken up and expressed by host cells, lea.ding to subsequent specific induction of both the humor-al and cell-mediated arms of the immune system. Significant levels of protection have been obtained using such an approach in a number of experimental animal systems. The Geneva meeting set out to assess the state of the art of nucleic acid vaccine technology, including an evaluation of its potential for developing new vaccines, and to review s,afety-related issues. It was organized into sessions on DNA vaccines, RNA vaccines, safety and regulatory issues, and vaccines currently being developed for specific diseases including malaria, hepatitis B, influenza, leishmaniasis, tuberculosis and HIV. DNA

Vaccines

Numerous routes for th,e delivery of DNA vaccines have now been tested. H. Robinson (University of Massachusetts Medical Center, Worcester, USA) presented data on DNA delivery by various procedures, including intramuscular, intravenous, intradetmal, intranasal and intraperitoneal routes. While the intraperitoneal route was found to be ineffective, all the others gave some response, with intramuscular being the most effective. A ‘gene-gun’, where DNA constructs on gold beads are ‘shot’ into the skin, was also reported to be a highly effective means of delivery. An effective DNA vaccine for influenza A in mice, based on the conserved, internal nucleoprotein, was reported by J. Ulmer (Merck Research Laboratory, West Point, USA). This vaccine, injected intramuscularly, induced 0

1994. Elsetier Science Ltd

both cytotoxic T lymphocyte (CTL) and antibody responses, and gave protection levels as high as 90% in challenge experiments in mice. Experiments to determine the duration of protection found that the level of protection had decreased partially by six months but could be restored to initial levels by reboosting. The CTL response was found to penist for at least a year. Studies to assess gene-transfer methods were reported by I. Danko (Univenity of Wisconsin, Madison, USA). A construct containing p-gal (the gene for /3-galactosidase) as a reporter, under control of a Rous sarcoma virus (RSV) promotor, was injected into exposed quadricep muscles. It was observed that some of the P-galactosidase-expressing cells were located distant from the injection site. The plasmid DNA was shown to exist in an open circular form and to retain a bacterial methylation pattern, indicating that it had not replicated. No evidence of integration was obtained. G. Rhodes (Vital Incorporated, San Diego, USA) described work which showed that vector sequences can affect the levels of protein expression. Using luciferase as a reporter, the relative levels of expression of various promoton including cytomegalovirus (CMV), RSV, and a muscle-specifc promotor, were compared. Relative to each other, the CMV promotor gave the highest levels of luciferase expression, being between two and three times higher than RSV, and, somewhat surprisingly, more than 20 times higher than the muscle-specific promotor. Injecting DNA in a hypertonic solution, such as sucrose, was reported by H. Davis (University of Ottawa, Canada) to increase expression, possibly by forcing the cells apart and tearing the inter-cell tissues, thereby enabling easier access to the host cells. The effectiveness of gene transfer into regenerating cells (pre-treated with cardiotoxin) was also described. Some of the potential advantages and liabilities of DNA vaccines were summarized by J. Ulmer. Advantages include the ability to generate specific CTLs without the potential risks of replicating vectors or live organisms, the fact that in situ expression of the antigen should relevant conformationally generate

epitopes, and that the technology may be more practical than production of recombinant proteins. Potential liabilities include the possibility of integration of the vector into host chromosomal DNA and the possible induction of tolerance or autoimmune responses (see below). RNA Vaccines Presentations in this session were made by P. Meulin (Pasteur-M&ieux S&urns and Vaccins, Marcy I’Etoile, France) and P. Liljestrom (Karolinska Institute, Huddinge, Sweden). The session began with an outline of some of the potential benefits of RNA vaccines. Unlike DNA, mRNA is transient in nature, does not persist in the cell and would not integrate into host chromosomal DNA. Work was presented in which RNA, encapsulated in liposomes, was able to induce specific CTL responses. Intravenous and subcutaneous routes were successful but, as with DNA delivery, the intraperitoneal route was not. Safety and Regulatory Issues Numerous safety and regulatory issues were discussed during the meeting. Invited speakers on safety and regulatory issues were H. Smith (Food and Drug Adminstration, Rockville, USA), K. Cichutek (Paul-Ehrlich Institute, Langen, Germany) and J. Robertson (National Institute for Biological Standards and Control, Potters Bar, UK). General guidelines that would apply to the assessment of a nucleic acid vaccine product include an analysis of its efficacy, potency, quality and purity, as well as its general safety and environmental risk assessment. Safety issues that would need to be assessed include a careful analysis of the effects of the various components used in the constructs themselves (including promotors, terminaton and selection markers), the possible generation of anti-DNA antibodies, and possible untoward consequences resutting from the persistence of the DNA and expression including tolerance, autoimmunity and hyperimmunity. Another important consideration is the possibility

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of an untoward transformation event occuring by integration of vector DNA into the host chromosome. An assessment of germ-line transfer should also be made. Specific Vaccines A promising DNA vaccine against murine malaria (Plasmodium yoelii) based on the circumsporozoite protein was reported by S. Hoffmann (Naval Medical Research Institute, Rockville, USA). High levels of protection (83%) were obtained in challenge experiments in mice and the vaccine was demonstrated to generate both specific antibody and CTL responses. Work on a DNA vaccine for hepatitis B virus was reported by R. Whalen (Pasteur Institute, Paris, France). Following vaccination of mice with a DNA construct encoding HBsAg, both the antigen itself and anti-HBsAg antibodies were detected. The concentration of antibody was significantly higher than that which is generally considered suitable to obtain protection in humans with the hepatitis B vaccine currently in use. Although it was noted that direct comparisons of antibody concentrations between mice and humans may not be meaningful, the data are, nevertheless, very promising. Chimpanzees have now been vaccinated in preparation for a challenge/protection experiment.

Experiments to develop a DNA vaccine for leishmaniasis, based on a highly conserved major surface glycoprotein, gp63, were described by F. Liew (University of Glasgow, UK). Preliminary experiments suggest that mice vaccinated with a construct containing the gene encoding gp63 develop smaller lesions than do control mice. A promising DNA vaccine against mycobactetia was reported by D. Lowrie (National Institute for Medical Research, London, UK). Mice injected with a DNA plasmid encoding the heat shock protein, hsp65, of mycobacteria developed substantial protection against challenge infection compared to control mice. The final speakers on specific vaccines presented data from experiments aimed at developing vaccines against human immunodeficiency virus (HIV). Presentations were made by A. Liu (Merck & Co. Inc., West Point, USA), D. Weiner (Hospital of the University of Pennsylvania, Philadelphia, USA), and K. Krohn (University of Tampere, Finland). Data presented in this session indicated that various constructs could induce both specific antibody and T-cell responses directed against HIV target antigens in both mice and monkeys.

IO,no. 12, I994

what action the General Progt-amme of Vaccination of the WHO could take to promote this technology further and direct its application to particular target diseases. Discussion was also held on the most appropriate term for this new technology, as several terms including nucleic acid vaccines, polynucleotide vaccines and genetic vaccines have been used to date. Among other things, it was felt that the term ‘genetic vaccine’ could be easily misconstrued. Following a lively discussion and subsequent vote, the overwhelming majority decided in favour of the term ‘nucleic acid vaccines’ being used in the future.

Acknowledgements The meeting on Nucleic Acid Vaccines, held at the World Health Organisation, Geneva, Switzerland, I7- I 8 May 1994, was organized within the scope of activities associated with basic vaccinology and vaccines for parasitic infections, by the WHO/UNDP Progtamme for Vaccine Development the UNDP/Wotid Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Global Programme on AIDS (GPA). Papers from the invited presenters at this meeting will appear in the November I994 issue of Vaccine.

Final Discussion The final discussion covered other areas, including patents, licensing and

Gory Waine is at the Tropical Health Program, Molecular Parasitology Unit Queensland Institute ofMedical Research, 300 Herston Road, Brisbane, Queensland 4029, Australia.

Molecular Parasitology in Scotland J, Sternberg and S. Graham

Kirkmichael, UK May 1994 For several years, there have been thrice-yearly afternoon symposia held, bringing together researchers in the various parasitology research groups in Scotland, and these became known as SUMP (Scottish Universities Molecular Parasitology) meetings. In 1993, SUMP held its first residential meeting and, in the wake of its success, in May l994,70 parasitologists gathered for a weekend of the latest advances in molecular and immunoparasitology. The meeting was held at Kindrogan House, a field studies centre set in an attractive part of the

Perthshire Highlands near Kirkmichael. Thanks to generous sponsorship for the second year, it was possible to invite an excellent group of guest speakers from the UK and overseas. Malaria: Genes and Chromosome Structure Jeff Ravetch (Sloan Kettering Institute, New York, USA) opened the proceedings with a review of the current research on malaria chromosome structure and the status of the malaria genome project. A fascinating dimorphism is becoming apparent in malaria chromosomes, in which it seems that

houskeeping genes tend to the central regions whereas antigen genes are found in sub-telometic regions. The subtelomeric regions are also recombinational ‘hot spots’. There is an active involvement in the malaria genome project in Scotland, and recent data on yeast artificial chromosome mapping of Plasmedium falciparum chromosome 6 was presented by Paul Meaney (Institute of Cell and Molecular Biology, Edinburgh, UK). Edinbutgh has long been a centre of excellence for the study of malaria genetics, and this was reflected in a series of contributions from the Institute of Cell Animal and Population Biology. Jane Cariton described the identification of a locus strongly linked to the chloroquine0

1994. Elsevier Science Ltd