Immature virus particles in dengue pathogenesis: Role of heterosubtypic immunity

Dengue is a rapidly emerging mosquito-transmitted viral infection and represents a major health problem in most of the tropical and sub-tropical regions of the world. There are four distinct serotypes of dengue virus (DENV 1 - 4) and each of these serotypes may cause disease ranging from mild febrile illness to devastating manifestations including Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS). DHF and DSS are a leading cause of hospitalization and death among of children in South-East Asia, Latin America and the Caribbean. There are no vaccines or antiviral treatments for DENV infection. The emergence of DHF/DSS has been investigated in considerable detail during the last three decades. Two groups of patients have been identified to be at risk for severe dengue disease: (i) individuals experiencing secondary infection with a heterosubtypic DENV virus and (ii) infants, born to DENV-immune mothers, undergoing a primary infection. Disease severity of DENV infection thus appears to be controlled primarily by pre-existing antibodies. The molecular mechanisms involved in antibody-dependent enhancement (ADE) of disease is still incompletely understood. Even though most of the antibodies against DENV in infected individuals are directed to the major viral envelope glycoprotein, E, it is known that sera of DENV-infected individuals may contain substantial levels of antibodies specific for the second viral membrane protein, prM. Furthermore, the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection, suggesting that anti-prM antibodies might be somehow involved in the development of severe disease. This is puzzling, because prM in fact is the precursor form of the envelope protein M, which is only present in immature virus particles that are generally considered to be non-infectious. Yet, DENV-infected cells secrete high numbers (~30%) of such immature prM-containing virions. How then could non-infectious immature virus and anti-prM antibodies contribute to the development of severe DENV infection and enhanced disease? Recently, we observed that immature DENV turns highly infectious in the presence of prM antibodies. It appears that, contrary to the normal viral maturation process which occurs during assembly in virus-infected cells, antibody-opsonized immature virus matures upon entry in new target cells. This observation suggests that immature DENV may indeed be involved in dengue pathogenesis. In this study, we will characterize in detail the infectious properties of prM-containing DENV particles and attempt to unravel the potential involvement of immature particles in dengue pathogenesis. Focus of the project will be on the role of different virus serotypes, since heterosubtypic immunity is a hallmark of ADE and severe disease. We will analyze both prM- and E-specific antibodies. In addition, in collaboration with the BIOMED institute of the University of Carabobo, Maracay, Venezuela, we will determine the proportion of immature virus particles that are present during a natural DENV infection. Finally, we will analyze patient sera for their ability to mediate ADE of DENV infection. Since virus-specific antibodies may have the capacity to either neutralize or enhance DENV infectivity, we anticipate that the results of this study will provide novel insights in disease pathogenesis that may have important repercussions for the development of a safe and efficacious vaccine against DENV infection.