Reserach Express@NCKU - Articles Digest

Dengue virus infection just like other infectious diseases has an iceberg characteristics. Most cases are symptomless, followed, in increasing rarity, by undifferentiated fever, dengue fever (DF), and life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DF/DHF is the most important arboviral disease of humans, occurring in tropical countries of the world including Taiwan. WHO has estimated that between 50 and 100 million cases of DF and several hundred thousand cases of DHF occur each year, depending on the epidemic activity. DHF is the leading cause of hospitalization for children in Southeastern Asia. Tainan also has a big dengue outbreak currently. Dengue virus infection causes two major clinical characteristics: thrombocytopenia and plasma leakage that are unique and distinct from other virus-caused diseases. Currently, no effective treatment except supporting care, and no approved vaccines are available.

The severe DHF/DSS mostly occur in secondary infection, especially when the second infection is by serotypes different from the previous one. This is an exception to the dogma of an established immune response can protect the subsequent infection. On the contrary, the previous immunity would enhance the disease process. Antibody-dependent enhancement (ADE) hypothesis is proposed to explain this phenomenon. The heterologous anti-dengue antibodies from previous infection or maternal derived will not neutralize the virus, instead, it would enhance the virus entrance into target cells via the Fc receptor after binding the dengue virion, thus leading to high virus burden and more severe disease development. The in vitro study indeed showed the enhancing activity of the anti-dengue IgG antibody. Epidemiological studies support the association of DHF/DSS with secondary dengue virus infection. However, this by itself explains neither the pathogenetic basis of the association nor the molecular mechanism of the DHF/DSS clinical manifestations. It is not clear how augmentation of dengue virus infection by enhancing antibodies leads to DHF/DSS, in addition to increase the viral mass. Will it enhance the dengue virus replication or suppress the anti-viral activity via FcRII signaling? What are the characteristics of the enhancing antibodies? Do the target cells require the receptors other than FcR? Which part or epitope of the dengue virion: E or prM is recognized by the enhancing antibodies? The mechanism of ADE has to be solved before we can generate the neutralizing activity without enhancement with the dengue vaccine candidate.

Using flow cytometric assay and monoclonal anti-dengue antibody, in this study, we observed that both anti-E and anti-prM Abs could enhance dengue virus infection in a concentration-dependent, but serotype-independent manner. Increases were found in both the percentage of dengue-infected cells and the expression of dengue E and NS1 protein per cell. Dengue virion binding and infection were enhanced on FcR-bearing cells via the Fc-FcRII pathway. Furthermore, anti-prM Ab also enhanced dengue virion binding and infection on cells lacking FcR, such as BHK-21 or A549 cells, by a peptide-specific binding. Anti-prM Ab cross-reacted with BHK-21 or A549 cells and recognized self-antigens such as heat shock protein 60. A novel mechanism of anti-prM Ab-mediated enhancement on dengue virus infection was found to be mediated by dual specific binding to dengue virion and to target cells, in addition to the traditional enhancement on FcR-bearing cells (Figure 1).

This finding has great impact on our understanding of dengue pathogenesis and the development of dengue vaccine. Using vaccine to prevent the infectious diseases is the art of modern preventive medicine, dengue vaccine has been tried for more than 50 years, but no commercial dengue vaccine is available yet. Some obstacles exist for this delayed development. An effective vaccine needs to prevent infection with all four dengue serotypes. Currently, several dengue vaccines have been undergoing the clinical trials. Most of them use the live attenuated virus or chimeric attenuated virus. One of the considerations using live vaccine is primarily to avoid the issue of ADE because subunit or inactivated vaccine preferentially stimulates antibody production that is much easily waning. In addition, the cell-mediated immunity post live vaccine immunization might be helpful in clearing the virus-infected cells and prevent or decrease the severity of DHF/DSS. However, the molecular mimicry between dengue virus prM and HSP60 self protein raises the concern of autoimmunity induction post dengue virus infection. The anti-prM antibody is not only an enhancing antibody, but also a pathogenic antibody and will enhance the target cell damage. The use of the live dengue vaccine will become problematic in the future. The induction of autoimmunity after live dengue virus immunization will post great concern not only for the safety, but also for the efficacy of the developing dengue vaccine.

For the dengue vaccine design, the live dengue virus or the whole inactivated dengue virus is not suitable as the vaccine candidate because of the molecular mimicry for prM, NS1 or NS3 to self proteins. The subunit E will be a preferred vaccine candidate target. The protection to dengue infection is largely mediated by the neutralizing antibodies. In dengue patients, the enhancing antibodies are simultaneously present with the neutralizing antibody, the enhancing activities were revealed only after the neutralizing antibodies were diluted out. The anti-dengue antibody from DF or DHF dengue patients can enhance the dengue virus infection in a concentration-dependent, but serotype-independent manner. We have to realize the difference between neutralizing antibody and enhancing antibody. The epitopes that stimulate the neutralization antibody has to be discriminated from those that induce the enhancing antibody. The epitope cross-reactive with the self protein needs to be identified and deleted. The E protein contains only the neutralizing epitopes, not the pathogenic or enhancing epitopes will be the dengue vaccine candidate. An idea dengue subunit E vaccine will be that it only induces the generation of the neutralizing activity, neither enhancing nor pathogenic autoantibody activity. This new concept of dengue vaccine design will challenge the currently developing live dengue vaccine candidates, and provide an opportunity to develop a safe and effective dengue vaccine in the future.