REPLICATION OF CHIMERIC YELLOW FEVER VIRUS-DENGUE SEROTYPE 1-4 VIRUS VACCINE STRAINS IN DENDRITIC AND HEPATIC CELLS SAMANTHA BRANDLER, NATHAN BROWN, THOMAS H. ERMAK, FRED MITCHELL, MEGAN PARSONS, ZHENXI ZHANG, JEAN LANG, THOMAS P. MONATH, AND FARSHAD GUIRAKHOO Institute of Virology, Medical University of Vienna, Vienna, Austria; Acambis, Inc. Cambridge, Massachusetts; Aventis Pasteur, Campus Merieux, Marcy-L’Etoile, France Abstract. ChimeriVax™-dengue (DEN) viruses are live attenuated vaccine candidates. They are constructed by replacing the premembrane (prM) and envelope (E) genes of the yellow fever (YF) 17D virus vaccine with the corresponding genes from wild-type DEN viruses (serotypes 1-4) isolated from humans. In this study, the growth kinetics of ChimeriVax™-DEN1-4 and parent viruses (wild-type DEN-1-4 and YF 17D) were assessed in human myeloid dendritic cells (DCs) and in three hepatic cell lines (HepG2, Huh7, and THLE-3). In DC, ChimeriVax™-DEN-1-4 showed similar growth kinetics to their parent viruses, wild-type DEN virus (propagated in Vero cells), or YF 17D virus (peak titers ∼3-4.5 log 10 plaque-forming units (PFU)/mL at 48-72 hours post-infection). Parent wild-type DEN-1-4 viruses derived from C6/36 mosquito cells did not show any growth at a multiplicity of infection of 0.1 in DCs, except for DEN-2 virus, which grew to a modest titer of 2.5 log 10 PFU/mL at 48 hours post-infection. ChimeriVax™-DEN1-4 grew to significantly lower titers (2-5 log 10 PFU/mL) than YF 17D virus in hepatic cell lines THLE-3 and HepG2, but not in Huh7 cells. These experiments suggest that ChimeriVax™-DEN1-4 viruses replicate similarly to YF-VAX in DCs, but at a lower level than YF 17D virus in hepatic cell lines. The lack of growth of chimeric viruses in human hepatic cells suggests that these viruses may be less hepatotropic than YF 17D virus vaccine in humans. INTRODUCTION Dengue (DEN) virus is a mosquito born flavivirus that in- fects 100 million people annually and causes 24,000 deaths in tropical and sub-tropical areas. There are four serotypes of DEN virus, all of which can result in severe forms of the disease known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Prior infection by one DEN virus se- rotype increases the likelihood of acquiring DHF/DSS upon exposure to a second DEN virus serotype. 1 Thus, the ratio- nale for successful vaccination requires simultaneous vaccina- tion against all four serotypes. ChimeriVax™-DEN1-4 is a tetravalent live attenuated vac- cine composed of four chimeric yellow fever (YF) 17D-DEN viruses. Each chimera was constructed by removing the pre- membrane (prM) and envelope (E) genes of the YF 17D virus, and replacing them with the genes of one of the four serotypes of DEN virus. 2 Because of the chimeric nature of these viruses, we were interested in comparing the replication and tissue tropism of the vaccine strains to the parental vi- ruses using dendritic cells (DCs) and hepatic cell lines. Myeloid immature DCs are found in most non-lymphoid organs, and form a network in the epidermis where they are also known as Langerhans cells. After capture and processing of a pathogen, immature DCs are activated and differentiate into mature DCs that migrate under the control of interleu- kin-1 to draining lymph nodes where they are responsible for antigen presentation, T cell activation, and stimulation of memory cells. 3,4 Langerhans cells are critical to the induction of immune responses to foreign antigens introduced into the skin. In a natural infection, DEN virus is introduced into the skin of the vertebrate host by the bite of an infected mosquito. Macrophages and monocytes were thought to be initial target cells for DEN infection, but recent studies have shown that DCs are 10-fold more permissive for DEN infection and rep- lication than either macrophages or monocytes. 5–8 The in- creased infectivity is facilitated by use of the DC-specific in- tercellular adhesion molecule 3-grabbing nonintegrin (DC- SIGN) receptor. 9,10 To our knowledge, there are no published reports showing replication of wild-type YF or YF 17D vac- cine viruses in DCs, although another flavivirus, West Nile virus, has been reported to replicate in DCs. 11 Due to the important role of DCs in initiating an immune response, we selected these cells for comparison of the growth kinetics of ChimeriVax™-DEN1-4 versus YF 17D virus as well as the four wild-type DEN virus serotypes (wild-type DEN1-4). We also addressed the hepatotropism of ChimeriVax™- DEN1-4 in hepatic cell lines. Yellow fever 17D and DEN viruses exhibit different patterns of hepatic infection. The YF 17D virus vaccine was developed in 1936 by empirical passage of wild-type YF virus in mouse and chick embryos. Although the vaccine virus has lost its viscerotropism and exhibited a marked reduction in neurovirulence properties for monkeys, there has been a number of serious adverse events (AEs) associated with this vaccine, resulting in encephalitis or he- patic failure. 12 During the course of DEN virus infection, the pathology of the liver is generally mild and resembles that of the early stages of YF virus infection, with less severe symp- toms such as limited lesions in the liver and mild foci of ne- crosis. The presence of DEN viral antigens in human hepa- tocytes from patients with dengue fever, demonstration of apoptosis in Kupffer cells, and productive replication in hu- man hepatoma cell lines (HepG2, Huh7) suggest that the liver can be targeted by DEN viruses. 13–19 Because severe hepatic failure with YF 17D virus (at a rate of  1:400,000) 20 and potential infection of the liver with DEN virus (DEN virus was recovered from 5 of 17 livers of children suspected to have died of dengue) 21 have been reported, the hepatotro- pism of the chimeric YF-DEN1-4 viruses for humans (in vivo) may only be revealed by monitoring vaccine-related AEs af- ter inoculation of large numbers of humans. To compare the hepatotropism of these chimeras to that of YF 17D and wild- type DEN viruses in vitro, we measured the growth kinetics of these viruses in three hepatic cell lines: HepG2, Huh7, and THLE-3. MATERIALS AND METHODS Cell lines. Normal human dendritic cells (NHDCs) were obtained from Cambrex Bio Science (Walkersville, MD). Am. J. Trop. Med. Hyg., 72(1), 2005, pp. 74–81 Copyright © 2005 by The American Society of Tropical Medicine and Hygiene 74