Dengue is the most rapidly spreading mosquito-borne viral disease in the world. In the last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries and, in the present decade, from urban to rural settings. An estimated 50 million dengue infections occur annually and approximately 2.5 billion people live in dengue endemic countries [1,2].
Since 2001, to support a 10-year plan to accelerate the introduction of safe and effective dengue vaccines, the Rockefeller Foundation and the Bill & Melinda Gates Foundation have provided funds to establish the Pediatric Dengue Vaccine Initiative (PDVI). PDVI has inaugurated a research programme targeted at understanding the mechanisms underlying the protection and enhancement of dengue virus infections and development, and the evaluation of new diagnostic tests for acute dengue infection. In addition, PDVI supports downstream development of selected dengue vaccine candidates.
In 2008, PDVI organized the first Pan-American Dengue Research Network Meeting in Recife, Brazil, under the theme “Epidemic dengue in the Americas: research challenges”. A second meeting has now taken place in Cancun, Mexico, 16–19th November 2010, co-sponsored by CONACYT (Mexico), CINVESTAV (Mexico), BIO-RAD (USA) and Standard Diagnostic Inc. (Korea). Around 200 scientists from Argentina, Australia, Brazil, Canada, Colombia, Cuba, Guatemala, México, Panama, Peru, Puerto Rico, Uruguay, USA, and Venezuela participated in a dynamic forum of scientific exchange that included plenary sessions, short talks, poster sessions and commercial exhibitions.
An extensive programme
In keeping with the current dengue situation, the meeting opened with a special lecture on dengue epidemiology in Mexico by Dr Celia Alpuche, Director of the Institute of Epidemiological Diagnosis and Reference (INDRE). The increasing burden of dengue in Mexico has been demonstrated by the number of cases, as well as the augmented ratio of DHF/DF. A key factor that has affected the quality of dengue surveillance in this country has been the insufficient number of diagnostic samples collected; most laboratories in Mexico (90%) have only IgM-based diagnostic tests, although 10% of the positive samples (and all samples from severe cases) are processed for virological surveillance. It was estimated recently that DENV-1 is predominant in Mexico, corresponding with 81.6% of isolations in 2010. A web-based dengue informative system for proper case notification is now needed, so that epidemiological data is available to facilitate early alerts.
The opening session then switched to basic science, with a special lecture on structure of dengue viruses/antibody complexes. Dr Felix Rey (Institute Pasteur, France) emphasized the implications of structure conservation on viral evolution, as well as the relevance of epitopes targeted by neutralizing antibodies for safety vaccines. The study of epitopes mapping conducted by Rey et al., support the role of Domain I in the envelope protein inducing strong neutralizing antibodies in humans, which could be relevant for future subunit vaccine candidates.
Subsequently, the meeting continued with an extensive programme covering progress in developing dengue vaccine candidates, host immune response, viral structure and cell entry, virus-host interaction, animal models/pathogenesis, viral replication and morphogenesis, clinical aspects of dengue disease, diagnosis, epidemiology, and vector biology.
Results of dengue vaccine clinical trials were presented by Sanofipasteur, NIH and Inviragen. Preclinical and clinical evaluations of the Sanofipasteur yellow fever-dengue chimera strategy support the favourable immunogenicity and short-term safety of this vaccine. Phase II clinical trials in endemic populations showed a balanced immune response to the four serotypes. Previous flavivirus infections seem to have priming potential. Results of the first worldwide efficacy study will be available in 2012. The NIH dengue 4–dengue chimeras and gene deletion strategies were evaluated in adults producing rash and neutropenia. The Inviragen DENVax tetravalent vaccine strategy –using attenuated DENV-2 PDK-53 virus and three chimeric recombinant viruses bearing the DENV-2 PDK-53 non-structural gene backbone expressing DENV-1, 3 or 4 structural genes – is currently being evaluated in two Phase I clinical trials.
During a session on third-generation dengue vaccines, results were presented on: DNA vaccines based on the dengue envelope protein and their combination with other vaccine strategy; the alphavirus replicon-based tetravalent dengue vaccine; the Cuban strategy based on the recombinant subunit vaccines using the envelope (Domain III of E protein fused to the P64k protein of Neisseria meningitides) and core proteins; and the GSK strategy using inactivated vaccine. This session included a roundtable for discussion of 2nd and 3rd generation vaccines. The absence of accepted correlates of protective immunity was discussed, as a key element required to achieve an adequate assessment of vaccine derived immunity. A better understanding of how interactions of virus with antibodies prevent target cell infection is also needed.
A special lecture on new insights into the molecular basis of neutralization by anti-dengue virus antibodies was presented by Michael Diamond (Washington University School of Medicine, St Louis, Missouri, USA). Characterization of around 300 new mouse monoclonal antibodies has revealed that protective monoclonal antibodies are localized to distinct epitopes in all three E protein domains and that neutralization potency correlates with a narrowed genotype and serotype specificity. Structural and functional complexity of antibodies with strong neutralizing activity was discussed.
Further studies on the structure of dengue virus antibody complexes were presented by Richard Kuhn (Markey Center for Structural Biology, USA). The stoichiometry of the viral structural components was evaluated by mass spectrometry. The structures of several assembly intermediates that contain the precursor PrM were established. Also dynamic movements of the virion were predicted, suggesting that the particle is not a static structure. This dynamic motion allows certain neutralizing antibodies to bind to normally cryptic sites on the E protein, preventing the protein transitions required for virus entry.
A special lecture concerning virus-host interactions was presented by Jorge Muñoz (Centers for Disease Control and Prevention, Puerto Rico) under the title “Modulation of the antiviral response by dengue virus”. Undoubtedly, the unveiling of these virus-host interactions leads to better understanding of dengue pathogenesis, and to innovative diagnostic and therapeutic approaches.
Clinical aspects and epidemiology of dengue in Brazil were discussed; an increase in disease severity has been seen here, mainly associated with children.
The session on animal models and pathogenesis was introduced by Eva Harris (University of Berkeley, USA) with a special lecture “Protective and enhancing potential of mouse and human natural and therapeutic antibodies”. These studies demonstrate that an AG129 mouse model reproduces lethal antibody-dependent enhancement (ADE) as well as antibody-mediated protection after DENV infection. Researchers found that modifications in human monoclonal antibodies prevent antibody dependent enhancement (ADE). Likewise, non-Fc?receptor-binding monoclonal antibodies are therapeutic after direct lethal challenge in mice. Importantly, the therapeutic efficacy seems to be more associated with the monoclonal antibodies characteristics than with the neutralizing antibody titres. This knowledge should provide insight into the role of antibodies in protection and pathogenesis of DENV infection in vivo.
Interesting presentations on replication and morphogenesis of DENV were then given in a session that included a special lecture “lipid droplets and viral morphogenesis” by Andrea Gamarnik (Foundation Leloir-CONICET Institute, Argentina). Little is known about the recognition of the viral RNA by the capside protein during genome encapsidation. Previous reports indicate that capside protein is associated with lipid droplets. Specific amino acids on the ? helix, located in the centre of the capside protein, have been found to be crucial for both accumulation of capside on lipid droplets and DENV infectious particle formation. It was proposed that lipid droplets play multiple roles during the dengue virus life cycle.
Some attractive research studies – including genetic studies of the mosquitoes and some studies supporting the immunological priming in Aedes aegypti – were presented during the vectors/ecology session. In addition, preliminary results were presented from the use of a strategy to produce a transgenic strain of Aedes aegypti which dies following infection with dengue virus, resulting in the elimination of transmission.
Perseverance needed
In addition to the special lectures and round tables, all sessions included several short talks, which (coupled with two poster sessions) summed up more than 100 presentations. Poster session I included several presentations on basic research concerning DENV entry and replication, genetic/immunological studies on dengue, and mechanisms involved in the host response to DENV. Poster session II focused mainly on diagnosis, molecular epidemiology, vector, and sylvatic hosts.
Notably, Brazil, Mexico and Cuba had the largest number of participants at the meeting. Specific groups – from FIOCRUZ, Universidade Federal de Minas Gerais, Universidade Federal de Rio de Janeiro, and the University of San Paulo (all from Brazil), CINVESTAV from Mexico and “Pedro Kouri” Tropical Medicine Institute from Cuba – demonstrated the advances they are achieving in dengue research.
During their concluding remarks, Rosa Maria del Angel (CINVESTAV, Mexico) and Scott Halstead (PDVI, USA) highlighted the quality of the meeting. They emphasized the necessity to persevere and work to unravel the complex mechanisms of dengue pathogenesis which are needed for good dengue vaccine designs.
This report has been written for TropIKA.net by Rosmari Rodriguez-Roche of the“Pedro Kouri” Tropical Medicine Institute, PO Box 601 Marianao 13, Havana, Cuba; email: rosmari@ipk.sld.cu.
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