Dengue vaccine development

264 NEWS AND ACTIVITIES In addition, the development of integrated pro- grammes to gather data should be considered, since those data obtained from police or health sources are often inadequate. Information should be exchanged on the design of measures that have been successfully executed, since this could help to avoid bias in pro- jected studies, improve statistical reliability, indicate useful precedents, and help in the choice of suitable controls. Risk assessment methodology should also be reviewed. Rehabilitation -Early identification of hazardous drinking patterns is vital; the drinking habits of individuals involved in accidents where alcohol consumption is a contri- butory factor should be modified by educational measures or treatment. -Alcohol education and counselling programmes at the workplace can help not only employees directly affected but also the general workforce, by reducing the number of accidents. Legal measures -Implementation and sustainment of changes in legislation should be linked with adequate enforce- ment and educational programmes. -Promotion of, and periodic local changes in the level of, police enforcement and prosecutions of drink/driving cases are essential. Such action should be supported by information programmes aimed at road users, and the implications of the results pro- duced should be discussed. -Legislation is needed to limit the commercial use of the media to present alcohol consumption as an element of a desirable life-style. -Reduction of alcohol availability in sports stadia, leisure grounds, and motorways should be encour- aged. -Introduction of probationary driving licences and/ or lower blood alcohol concentration limits should be considered for inexperienced or learner drivers. -Random tests to detect drink/driving should be promoted. Education -Initiatives should primarily involve local com- munities, while at a secondary level the police, judiciary, drink trade, insurance companies, edu- cation and health authorities should participate. -The public should be informed of the high cost to society of alcohol-related accidents, since the perception by individuals of their risk is low. -The effectiveness of campaigns is markedly in- creased if harmonization is achieved between the various bodies involved. In this respect, straight- forward messages such as "Don't drink and drive" should be advocated and repeated regularly. Dengue vaccine development As part of the WHO Programme for Vaccine Development, a Steering Committee on Dengue was established in 1984 and charged to bring about as rapidly as possible the availability of vaccines that would be effective against all four serotypes of dengue virus. The task of developing a vaccine against Japanese encephalitis, a major health prob- lem in Asia caused by a flavivirus that is serologically and biophysically related to the dengue (DEN) viruses, was added in 1986 to the Committee's responsibilities. The Committee has encouraged laboratory studies aimed at producing second- generation vaccines using biotechnology. At the same time, progress has been monitored on the develop- ment of attenuated DEN vaccines against all four serotypes, a major project being conducted at Mahi- dol Universc .y, Bangkok, with WHO support. The Stewring Committee has prepared a strategic plan on thv, biotechnological approach to the pro- duction of second-generation vaccines, dealing with identification of protective epitopes, their expression, and a molecular definition of virulence. The plan provides general guidelines on achieving the goal of immunization against dengue and serves as a guide for evaluating proposals for financial support through WHO. For the purpose ofbringing togetherWHO grantees and other virologists who are making a contribution to the development of flavivirus vaccines, WHO sponsored a meeting on Current Approaches for the Development of Dengue Vaccines and Related As- pects of the Molecular Biology of Flaviviruses, which was organized in conjuncton with the VII Inter- national Congress of Virology, Edmonton, Canada, 7-8 August 1987. The results of human trials with a live attenuated DEN-2 virus candidate vaccine were reviewed at the meeting. This vaccine has been used to immunize 42 flavivirus-seronegative adult volun- teers, with the induction of significant levels of neutralizing antibodies. A live attenuated DEN-1 vaccine is now undergoing phase-I trials in adult volunteers. Encouraging results were presented at the meeting on the protective effect of immunization with the virus-specific nonstructural protein NS1. The interest in this protein as a vaccine arises because NS 1 antibodies do not react with the virion and therefore do not present the risk of enhanced infection of Fc receptor-bearing cells, which are associated with most cases of dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). An effective vaccine may well include both NS1 and an epitope from the envelope glycoprotein (E) that induces protective but not infection-enhancing antibodies. Studies to map the epitopes on E as well as NEWS AND ACTIVITIES 265 their expression have therefore been encouraged by WHO to identify those that afford such protection. Epitopes that exhibit various degrees of specificity for one or more flaviviruses as well as different reactivities in the serological and functional tests have been identified using monoclonal antibodies. Studies to fingerprint oligonucleotides in genomic RNA have been performed on many strains of DEN-1, DEN-2, and DEN-3 viruses and the data examined by computer analysis. The pattern for each serotype is distinct and reproducible, and similar fingerprints were obtained on virus genomes from a single serotype isolated during an epidemic in a particular geographical area. Thus far, 15 genotypic variants or topotypes of DEN-2 virus have been established, seven ofDEN- 1, and five ofDEN-3. The genomes of many strains of dengue virus and other flaviviruses are now being sequenced and this might provide some insight into the differences between virulent and avirulent or attenuated strains. The organization of the genome of dengue virus is similar to that of other flaviviruses, with the following order of viral genes from the 5'-end: C, prM, M, E, NS1, ns2a, ns2b, NS3, ns4a, and NS5. Several laboratories have now succeeded in expressing different viral genes in eukaryotic or prokaryotic systems and the products are being studied as potential immunogens. The nucleotide sequences of the virulent Asibi and the 17D strains of yellow fever virus have been compared and several base changes identified, al- though their relation to the attenuated phenotype has not yet been established. Similar comparative sequencing is currently being carried out with parental-vaccine pairs of dengue virus. The need for an information network for flavivirus nucleotide sequences was expressed during the meeting. Such a network could be coordinated by one of the WHO Collaborating Centres for Arbovirus Reference and Research under the coordination of the Steering Committee. There was concern about the lack of an appropriate animal model to evaluate candidate dengue vaccines, and the use of approved adjuvants in enhancing the immunogenicity of pep- tides was also discussed. At present, the flavivirus envelope protein that has been expressed is immuno- genic but has not yet been proved to be protective. Expression of the envelope glycoprotein in its native form is important, inter alia, for the assessment of protective immunity and for X-ray crystallographic studies. The next important milestone that will further the goal of genetically engineered flavivirus vaccines will be the production of infectious DNA clones. WHO will continue to provide a forum for spe- cialists to exchange information for the purpose of promoting and using new concepts in the production of vaccines against flaviviruses. Safety requirements for contraceptive steroidse One of the major objectives of WHO's Special Programme of Research, Development, and Research Training in Human Reproduction is to promote and support research to find new, safe, and effective methods of fertility regulation. Since its inception in 1971, the Special Programme has therefore moni- tored, inter alia, the testing of contraceptive steroids. Studies have revealed that there are major differ- ences in the reproductive physiology, and hence in the responsiveness of target organs to different steroidal drugs, between humans and laboratory test animals. Also, the results of epidemiological investi- gations indicate that, because of these differences, it is impossible to reliably predict the effects of contra- ceptive steroids in humans based on the results of animal studies alone. These findings have highlighted the importance of carrying out meticulous early studies in humans and careful and prolonged post-registration surveillance of contraceptive steroidal drugs. Furthermore, the International Conference on Population, held in Mexico City in August 1984, unanimously passed a resolution which included the recommendation that modernization and updating of the official require- ments for the preclinical and clinical assessment of new fertility-regulating agents and a strengthening of the research capabilities of developing countries in these areas are also urgently needed. In response to this, a Symposium on Improving Safety Requirements for Contraceptive Steroids was convened by the Special Programme in Geneva, 2-5 February 1987. Participants included basic and clini- cal scientists, as well as representatives of drug regulatory agencies, the pharmaceutical industry, and consumer groups. The major issues discussed were: -How predictive have animal studies been on the safety of contraceptive steroids in the light of sub- sequent epidemiological findings? -What measures could be taken to improve the validity of the animal models used in toxicological studies? -What improvements could be made to the design and organization of clinical trials of new contra- ceptive steroids? -What should be done to ensure the identification of long-term sequelae or rare adverse outcomes that may be associated with new contraceptive steroids? ' Based on: Guidelinesfor the toxicological and clinical assess- ment and post-registration surveillance of steroidal contraceptive drugs. Issued by the WHO Special Programme of Research, Development, and Research Training in Human Reproduction, document number HRP/SP.REP/87. 1. NOTES ET ACTIVITtS 275 -On peut sensiblement amnliorer 1'efficacite des campagnes en harmonisant I'action des diff6rents organismes qui y participent. A cet egard, des mes- sages simples tels que "boire ou conduire, il faut choisir" devraient etre inlassablement repet6s. Mise au point de vaccins contre la dengue Dans le cadre du Programme OMS pour la mise au point de vaccins, il a ete cre en 1984 un Comite d'orientation sur la dengue charge de hater la mise au point de vaccins efficaces contre les quatre serotypes du virus de la dengue. En 1986, on a ajoute a ses attributions la mise au point d'un vaccin contre l'encephalite japonaise, maladie due a un flavivirus serologiquement et biophysiquement apparente au virus de la dengue (DEN), et qui constitue un serieux probleme de sante en Asie. Le comite a encourage les etudes de laboratoire visant a produire des vaccins de seconde generation par des methodes biotechno- logiques. Dans le meme temps, il a suivi les progres d'un important projet de mise au point de vaccins attenues contre les quatre serotypes de virus de la dengue, projet qui est en cours de realisation a la Mahidol University de Bangkok, avec le soutien de l'OMS. Le comite d'orientation a defini une strategie d'utilisation de la biotechnologie pour la production des vaccins de seconde generation, qui comprend l'identification des epitopes protecteurs, leur ex- pression et la definition moleculaire de la virulence. Le plan etabli a cet effet trace les grandes lignes de la voie a suivre pour mettre au point un vaccin contre la dengue et sert de guide pour evaluer les demandes de financement adressees a l'OMS. Afin de favoriser les echanges entre les chercheurs bendficiant d'une bourse de l'OMS et les autres virologistes qui s'int6ressent a la production de vaccins contre les flavivirus, l'OMS a parraine une reunion sur les approches actuelles en matiere de mise au point de vaccins contre la dengue et les aspects con- nexes de la biologie mol6culaire des flavivirus. Cette reunion, organisee a l'occasion du ViHe Congres international de Virologie, qui a eu lieu a Edmonton, au Canada, les 7 et 8 aodt 1987, a passe en revue les resultats des essais effectues chez l'homme avec un vaccin experimental prepare a partir du virus DEN-2 vivant attenue. Ce vaccin a ete administre a 42 volontaires, seronegatifs pour les flavivirus, et il a induit des concentrations importantes d'anticorps neutralisants. Un vaccin vivant attenue DEN-1 fait egalement l'objet d'essais de phase I chez des volontaires. Des resultats encourageants ont ete presentes a la reunion en ce qui concerne 1'efficacite de l'immuni- sation par la proteine fonctionnelle NS1 specifique du virus. L'intrt de cette prot6ine comme vaccin vient de ce que les anticorps anti-NSI ne r6agissent pas avec le virion, de sorte qu'ils ne risquent pas de renforcer l'infection des cellules porteuses des recepteurs du fragment Fc, que l'on retrouve dans la plupart des cas de dengue hemorragique ou de syndrome de choc associe a la dengue. Un vaccin efficace pourrait comprendre a la fois la proteine NS1 et un epitope de la glycoproteine d'enveloppe (E) induisant des anticorps protecteurs, mais ne renforcant pas l'infection. L'OMS a donc encourage les etudes portant sur la cartographie des epitopes de cette glycoprot6ine E et sur leur expres- sion, en vue de rep6rer ceux qui offrent une telle protection. Grace aux anticorps monoclonaux on a identifie des epitopes qui pr6sentent divers degres de specificite vis-a-vis d'un ou de plusieurs flavi- virus, et des reactivites differentes dans les epreuves serologiques et fonctionnelles. On a procede a la cartographie des oligonucleotides de l'ARN gdnomique sur de nombreuses souches de virus DEN-1, DEN-2 et DEN-3 et les donnees ob- tenues ont ete analysees par ordinateur. Le profil de chaque s6rotype est distinct et reproductible et on a obtenu des cartes genomiques semblables pour les virus provenant d'un meme serotype isole au cours d'une epid6mie dans une region geographique determin6e. Jusqu'ici, on a identifie 15 variants genotypiques ou topotypes du virus DEN-2, sept du virus DEN-1 et cinq du virus DEN-3. On est en train d'etablir la sequence genomique de nombreuses souches de virus de la dengue et d'autres flavivirus, ce qui pourrait donner un apercu des differences qui existent entre les souches virulentes et les souches non virulentes ou attenu6es. L'organisation du g6- nome du virus de la dengue est semblable a celle des autres flavivirus, et presente les genes viraux dans l'ordre suivant, a partir de l'extr6mite 5': C, prM, M, E, NS1, ns2a, ns2b, NS3, ns4a et NS5. Plusieurs laboratoires ont maintenant reussi a exprimer diffe- rents genes viraux dans des systemes eucaryotes ou procaryotes et l'on est en train d'etudier les proprietes immunogenes eventuelles des produits obtenus. Les sequences nucleotidiques de la souche viru- lente Asibi et de la souche 17D du virus de la fi6vre jaune ont ete comparees et l'on a observe plusieurs modifications au niveau des bases, mais le lien avec I'attenuation de la virulence du phenotype n'a pas encore 6t6 dtabli. Un sequencage comparatifdu m6me type est actuellement en cours sur les souches d'ori- gine et les souches vaccinales du virus de la dengue. Au cours de cette reunion, le besoin d'un reseau d'information sur les sequences nucleotidiques des flavivirus s'est fait sentir. Un tel reseau pourrait etre 276 NOTES ET ACTIVITtS coordonne par l'un des centres collaborateurs de 1'OMS pour les arbovirus, sous la responsabilite du Comite d'orientation. On s'est inquiet6 du manque de modeles animaux appropries pour 6valuer les vaccins exp6rimentaux contre la dengue, et l'on a egalement discute de l'emploi d'adjuvants approuv6s pour renforcer l'immunogenicite des peptides. Actuelle- ment, la prot6ine d'enveloppe des flavivirus qui a ete exprimee est immunogene, mais ne s'est pas encore revelee protectrice. I1 est important d'exprimer cette glycoprot6ine d'enveloppe sous sa forme naturelle, notamment pour evaluer I'immunite protectrice qu'elle induit et pour faire des etudes de cristallo- graphie par diffraction des rayons X. La prochaine etape cruciale dans la mise au point des vaccins contre les flavivirus obtenus par genie g6n6tique sera la production de clones d'ADN infectieux. L'OMS continuera a servir de lieu de rencontre pour les sp6cialistes d6sireux d'echanger des infor- mations concemant la promotion et l'utilisation de nouvelles methodologies de production de vaccins contre les flavivirus. Normes d'innocuite des contraceptifs steroidiense L'un des principaux objectifs du Programme sp6cial OMS de recherche, de developpement et de formation a la recherche en reproduction humaine est de pro- mouvoir et de soutenir les recherches visant a mettre au point des methodes nouvelles, sfires et efficaces de r6gulation de la f6condit6. Depuis sa creation en 1971, le Programme special s'emploie donc, entre autres activites, a contr6ler les essais de contraceptifs steroidiens. Des etudes ont revel qu'il existait des differences majeures en matiere de physiologie de la repro- duction, et donc dans la facon dont les differents organes cibles reagissent aux medicaments steroi- diens, entre la femme et les animaux de laboratoire. De meme, les resultats des etudes 6pid6miologiques indiquent qu'en raison de ces differences, il est impossible de predire avec fiabilite les effets des contraceptifs steroidiens chez la femme uniquement sur la base des r6sultats obtenus avec des animaux de laboratoire. Ces constatations ont montre qu'il etait important d'effectuer des etudes meticuleuses chez la femme mais aussi d'exercer une surveillance rigoureuse et prolong6e des contraceptifs st6roidiens apres leur e D'apres: Guidelines for the toxicological and clinical assess- ment and post-registration surveillance of steroidal contraceptive drugs. Document pr6par6 par le Programme spdcial OMS de re- cherche, de dcve[oppement et de formation 'a la recherche en reproduction humaine sous la cote HRP/SP.REP/87.1 (en anglais seulement). enregistrement. De plus, la Conference internationale sur la Population, qui s'est tenue a Mexico en aoit 1984, a adopte a l'unanimit6 une resolution recom- mandant d'actualiser et de mettre a jour la regle- mentation officielle touchant l'6valuation pr6clinique et clinique des nouveaux agents de regulation de la fecondite et de renforcer les capacites de recherche des pays en developpement dans les domaines sus- mentionnes. En application de cette resolution, le Programme special a organis6 a Geneve, du 2 au 5 fWvrier 1987, un symposium sur l'am6lioration des normes rela- tives a l'innocuit6 des contraceptifs steroidiens. Y ont participe des sp6cialistes de la recherche fondamen- tale et clinique, ainsi que des repr6sentants des organismes de reglementation pharmaceutique, de l'industrie pharmaceutique et des associations de consommateurs. Les principales questions qui ont et6 pos6es ont WtE les suivantes: -Quelle a ete la valeur pr6dictive des etudes realis6es chez l'animal en ce qui concerne l'innocuite des contraceptifs st6roidiens, compte tenu des resultats des 6tudes epid6miologiques ulterieures? -Quelles mesures pourrait-on prendre pour ame- liorer la validite des modeles animaux utilis6s dans les etudes toxicologiques? -Quelles ameliorations pourrait-on apporter a la conception et a l'organisation des essais cliniques de nouveaux contraceptifs steroidiens? -Que faudrait-il faire pour deceler les eventuels effets secondaires a long terme ou les rares reactions indesirables qui peuvent etre associees aux nouveaux contraceptifs steroidiens? En r6sume, les principales conclusions formul6es par le symposium ont ete les suivantes: -Les effets chez l'animal des contraceptifs steroi- diens n'ont donn6 que peu d'indications sur certains effets secondaires qui ont ete deceles par la suite dans le cadre de recherches epidemiologiques. Les parti- cipants ont donc souligne la n6cessite de rationaliser les methodes et normes d'essai existantes, notamment en ce qui conceme les essais chez l'animal. A cet egard, il a ete recommand6 que les modeles animaux soient selectionnes sur la base des caract6ristiques pharmacocinetiques et pharmacodynamiques du medicament faisant l'objet de l'exp6rimentation. -II y a de bonnes raisons de mettre en doute l'utilit6 de certaines des normes relatives aux etudes de cance- rogenicite a long terme des contraceptifs steroidiens, par exemple, les etudes de 7 ans sur la chienne beagle et les etudes de 10 ans sur des primates. -Bien que la r6glementation actuelle concernant les