Halstead and colleague Srisakul (Susie) Kliks, PhD, PDVI's Deputy Director of Supportive Research, emphasized that milestones in dengue are as much about understanding the pathogenesis of dengue virus infection and dengue vaccineinduced immunity as they are about licensing a vaccine, which Kliks, who is also Director of the Vector Borne Diseases Research Program at the School of Public Health at University of California Berkeley, predicts will be assisted by the development of correlates of immunity to accelerate Phase III studies. These may also support crucial postlicensing Phase IV studies that are likely to extend into the distant future.

Halstead points to six major highlights for PDVI and dengue study over the past year that exemplify PDVI's wide range of research. However, he is quick to point out that "even though some of these accomplishments have been recognized or come to fruition in the past 12 months, they are the results of years of effort on the parts of many scientists and researchers."

What is it? The envelope (E) protein of dengue virus plays a key role in viral infection both on the surface and inside the target cells. Understanding the structure of the envelope provides strategic targets for intervention in viral infection through vaccines or drugs.

Why is it important? The structure and function of the viral envelope are critical to successful intervention via a safe and effective vaccine. For example, PDVI-supported research revealed in the past year that the virus is internalized from the cell surface into a vesicle inside the cell. The subsequent fusion between the viral membrane and the vesicle provides an ideal target for intervention. These findings were substantiated by another group in the network that succeeded in directing a specific antibody against a conformational site of the envelop protein and thereby preventing fusion and aborting the infection.

What are the implications? The antibody directed to this specific site on the viral envelope can be used therapeutically to control viral infection and minimize clinical severity. In addition, the portion of the envelope that is the target of this specific antibody can be "re-tooled" and used as a component for an effective vaccine against the virus.

What is it? A mouse model for dengue virus infection. Several PDVI-sponsored groups working with dengue in mice determined that the mothers can transfer antibodies via the placenta to unborn offspring. This gives the infant mice a base level of dengue antibodies - a possible health hazard since certain levels of antibody may lead to enhanced infection when the individual is exposed to a dengue virus. The group took on the challenge of showing that the mice suffered severe disease in the presence of low amounts of antibodies. Large amounts of antibodies are protective. Using the mouse model, researchers can measure levels of antibodies for their protective effectiveness. The work is under review for publication by Nature.

Why is it important? Currently, there is no fully-functional animal model for dengue illnesses. An animal model that develops a disease very similar to dengue hemorrhagic fever is required to evaluate candidate dengue vaccine efficacy during the pre-clinical vaccine development stages.

What are the implications? An animal model will allow researchers to evaluate interventions, including a vaccine, in vivo, as well as better understand the process of dengue infection and possible acquisition of infection and to test the relation of antibody levels to immune status.

What is it? One of the largest collaborative autopsy studies on dengue fever. PDVI spearheaded this effort in Myanmar. The tissues are collected from children who have died of dengue hemorrhagic fever and are frozen or fixed in formalin. They are transferred to Siriraj Medical School in Bangkok and infected cells are identified. These cells are analyzed to prove that dengue virus infection occurred and which of the four types of dengue was involved.

Why is it important? As dengue treatments improved over the years, patients with suspected dengue were not routinely autopsied when they died. As a result, causes of death were not clearly determined or tracked and progress was slowed in understanding how dengue viruses caused illnesses in humans.

What are the implications? Improvements in dengue vaccine design and anti-dengue treatments can be expected to grow out of an improved understanding of dengue infection and host response at a cellular and molecular level.

What is it? A form of photography that allows research scientists to watch the entire process of infection from the time that a virus particle attaches to a cell, through cell entry, and to infection. This includes imagery of entry into endosomal compartments, fusion with the endosomal membrane and release into the cytoplasm, which leads to the full-blown infection.

Why is it important? Watching the entire process of infection may give researchers new insight into how the infection actually takes place, where it is initiated, and how to stall or prevent it entirely. For example, recently it was discovered that an experimental antibody that appears to cure dengue infections does so by preventing the fusion of the virus with the endosomal membrane. This resulted in the visualization of the blockade of dengue virus fusion by antibodies via confocal microscopy.

What are the implications? Combining this imagery with the laboratory work mentioned previously will provide important insights into the process of dengue infection. If the attachment molecules on the surface of human monocytes and macrophages that permit dengue entry without antibodies are identified, a successful dengue vaccine may prevent attachment at these sites.

What is it? Studies on how dengue interacts and infects cells. Two PDVI research groups are tracking the molecular processes of dengue infection in vitro. They have shown that when a dengue virus coupled to an antibody infects, it attaches to a specialized target cell by targeting a receptor on the surface of monocytes and macrophages and transmits a specialized signal to the cell to switch off certain immune responses (type 1 interferon production) so that the virus can take over.

Why is it important? Understanding how the virus invades target cells in the absence and presence of dengue antibodies will be vital to future vaccine development as well as to provide insights into the intracellular mechanisms of infectious diseases.

What are the implications? Once the specific process of dengue infection is known, researchers can use that information to devise antiviral therapies and preventative vaccines to interrupt the process. The results of the study and the details of the process are currently submitted for publication.

What is it? An ongoing PDVI-sponsored research initiative that tracks thousands of children in three geographical sites where all four dengue viruses can be found: Managua, Nicaragua, and Ratchaburi and Kamphaeng Phet provinces in Thailand. These children may be infected once, twice, or even more often with dengue viruses over the course of their lives. Each year, the group collects blood samples before and after the dengue transmission season, then uses antibody studies to determine how many children are infected over the course of a year, and with which type of dengue.

Why is it important? Information learned from this research can be used to directly identify a tendency to develop, resist, or succumb to dengue virus. The study includes sera from children who experienced mild or severe dengue infections and "silent" dengue infections that may have partial immunity to the infection. Cohort studies also provide accurate data on the illnesses that accompany dengue infections. This information can be used to make accurate national estimates on the clinical burden and cost of dengue illness.

What are the implications?This research is being used to collect "Gold Standard Sera" that can be used to validate tests for the evaluation of vaccines based on which patients became ill and which resisted the infection effectively.