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Am. J. Trop. Med. Hyg., 72(1), 2005, pp. 74-81
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

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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
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ChimeriVaxTM-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 ChimeriVaxTM-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, ChimeriVaxTM-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 log10 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 log10 PFU/mL at 48 hours post-infection. ChimeriVaxTM-DEN1-4 grew to significantly lower titers (2–5 log10 PFU/mL) than YF 17D virus in hepatic cell lines THLE-3 and HepG2, but not in Huh7 cells. These experiments suggest that ChimeriVaxTM-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
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dengue (DEN) virus is a mosquito born flavivirus that infects 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 serotype increases the likelihood of acquiring DHF/DSS upon exposure to a second DEN virus serotype.1 Thus, the rationale for successful vaccination requires simultaneous vaccination against all four serotypes.

ChimeriVaxTM-DEN1-4 is a tetravalent live attenuated vaccine composed of four chimeric yellow fever (YF) 17D-DEN viruses. Each chimera was constructed by removing the premembrane (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 viruses 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 interleukin-1ß 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 replication than either macrophages or monocytes.5–8 The increased infectivity is facilitated by use of the DC-specific intercellular 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 vaccine 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 ChimeriVaxTM-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 ChimeriVaxTM-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 hepatic 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 symptoms such as limited lesions in the liver and mild foci of necrosis. The presence of DEN viral antigens in human hepatocytes from patients with dengue fever, demonstration of apoptosis in Kupffer cells, and productive replication in human 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 hepatotropism of the chimeric YF-DEN1-4 viruses for humans (in vivo) may only be revealed by monitoring vaccine-related AEs after 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines. Normal human dendritic cells (NHDCs) were obtained from Cambrex Bio Science (Walkersville, MD). These cells were derived from peripheral blood mononuclear cells that were purified by leukopheresis, depleted of non-adherent cells, and cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and inter-leukin-4 (IL-4). Cambrex Bio Science NHDCs were reported to express low to absent levels of CD3, CD14, and CD83, and high levels of CD1a, CD11c, and CD86 as determined by flow cytometry. For infection studies, cells were thawed and cultured for 24 hours in T25 flasks or 12-well plates at a density of 3.4 x 104 cells/cm2 in lymphocyte growth medium 3 (Cambrex Bio Science), and supplemented with recombinant human IL-4 and recombinant human GM-CSF (R&D Systems, Minneapolis, MN) at a final concentration of 500 units/mL. Immature DCs (iDCs; obtained from a patient with acute myeloid leukemia AML) were obtained from NEMOD Immuntherapie AG (Berlin, Germany). This cell line represents precursor DC (prec-NemodDCTM, which can differentiate into immature DCs (i-NemodDCTM) with all the phenotypic and functional characteristics typical of human DCs. The iNemodDCs are reported to express high levels of major histocompatibility class I, class II, CD1a, and CD209 (DC-SIGN), and low to absent levels of CD83 and CD86. The iDCs were cultured in AIM V medium (Invitrogen, Carlsbad, CA) and plated into 12-well plates four hours prior to infection at a density of 5 x 104 cells/cm2.

The HepG2 and THLE-3 cells were obtained from American Type Culture Collection (Manassas, VA). The Huh7 cells were provided by Dr. Mike Holbrook (University of Texas, Medical Branch, Galveston, TX). Vero cells were obtained from the Acambis Cell Culture Facility and used in plaque assays for titration of viruses.

Viruses. Wild-type DEN1 (strain PUO 359, Thailand) (TVP-1140) was passaged once in C6/36 cells (mosquito derived virus, titer = 1 x 106 plaque-forming units [PFU]/mL) and four times in Vero cells (mammalian-derived virus, titer = 6.15 x 106 PFU/mL); wild-type DEN2 (strain PUO 218, Thailand) was passaged twice in C6/36 cells (titer = 1.2 x 107 PFU/mL) and three times in Vero cells (titer = 5.05 x 105 PFU/mL); wild-type DEN3 (strain PaH881/88, Thailand) was passaged once in C6/36 cells (titer = 3.6 x 107 PFU/mL) and four times in Vero cells (1.6 x 105 PFU/mL); and wild-type DEN4 (strain 1228, Indonesia, TVP 980) was passaged twice in C6/36 cells (titer = 4.6 x 105 PFU/mL) and four times in Vero cells (titer = 3.6 x 106 PFU/mL). ChimeriVaxTM-DEN1-4 viruses (titers = 4.63 x 107, 2.4 x 107, 1.4 x 106, and 4.4 x 106 PFU/mL, respectively) were passaged 10 times (vaccine level viruses produced from current good manufacturing practices [cGMP] Master Seed stocks [eight passages] in Vero cells).1 Commercial YF 17D virus vaccine, YF-VAX®, was obtained from Aventis Pasteur (Swiftwater, PA) and was used unpassaged (titer = 3 x 105 PFU/mL).

All experiments with chimeric viruses in hepatoma cells (Huh-7, HepG2, and THLE3) were performed at a multiplicity of infection (MOI) of 0.001. This MOI was selected based on previous growth curve experiments in Vero cells used for GMP manufacture of these viruses. The rationale for using a low MOI was to exclude a subpopulation of quasi-species viruses, which may have been present in the virus preparations not detected by consensus sequencing (which has a limit of detection of a mutation of about 10%). For experiments in DCs, we intended to use an MOI higher than 0.001 to be consistent with other investigators who used high MOIs (e.g., 5 or 50).10 The titer of commercial YF-VAX of ~3 x 105 PFU/mL did not allow us to go beyond the MOI of 0.1 for this virus. To perform the test at a similar MOI for all viruses, we therefore adjusted the MOIs of the chimeras to that of YF-VAX, which had the lowest titer. To exclude the possible prozone effect, which has been observed with the use of YF-VAX (e.g., earlier viremia, lower immunogenicity with undiluted vaccine)22,23 and might be due to the presence of crude chick embryo lysate or other excipients, YF-VAX was used at 1:2 or 1:4 dilutions without any further cell culture passages.

Infection of DCs. The DCs were grown in T25 flasks or 12-well plates at a density of 3–5 x 104 cells/cm2 and infected at an MOI of 0.1. The DCs were infected with wild-type DEN1-4 passaged in C6/36 mosquito cells or in Vero cells, ChimeriVaxTM-DEN1-4, and YF 17D viruses. After incubation for two hours at 37°C in an atmosphere of 5% CO2, 7.5 mL/T25 flask or 1.2 mL/well (12-well plates) of cell culture medium was added (since DCs were found to be sensitive to manipulations, cells were not washed to remove inocula) and cells were incubated at 37°C in an atmosphere of 5% CO2 for four days. To demonstrate that the amount of virus present in the DC supernatants represents the DC virus output and not the virus inoculum, a virus control, at the same concentration as in the DC experiments, was incubated in tissue culture plates in the absence of DCs. This was especially important in experiments where the virus output was not significantly higher than the inoculum used for DC infection. The DC supernatant samples and their controls (0.5 mL or 0.075 mL) were collected at 0, 2, 8, 24, 48, 72, and 96 hours post-infection for the growth kinetic studies. Since DCs are loosely adherent, samples were centrifuged and pellets were resuspended in 0.5 mL or 0.075 mL of cell culture medium and returned to the corresponding flask or well. The cell-free supernatants were stored in 50% heat-inactivated fetal bovine serum (FBS) at –80°C to preserve potency.

Infection of hepatic cells. The HepG2, Huh7, and THLE-3 cells were grown to confluency in T25 flasks at 37°C in an atmosphere of 5% CO2. The HepG2 cells were grown in Eagle’s minimal essential medium (MEM) supplemented with 8% FBS (ATCC, Manassas, VA) and 1% Antibiotic/Antimycotic (Sigma, St. Louis, MO). The Huh7 cells were grown in Eagle’s MEM (Gibco-BRL, Gaithersburg, MD) supplemented with 8% heat-inactivated FBS (Hyclone, Logan, UT) and 1% Antibiotic/Antimycotic (Sigma). The THLE-3 cells were grown in the bronchial epithelial basal medium media kit (Clonetics, San Diego, CA) without the addition of epinephrine and gentamicin/amphotericin components) and supplemented with 8% heat-inactivated FBS (Hyclone). The HepG2 (85 passages), Huh7 (44 passages), and THLE-3 (four passages) cells were infected with wild-type DEN1-4 (passaged in C6/36 cells), ChimeriVaxTM-DEN1-4, and YF 17D viruses at an MOI of 0.001 for one hour. Cells were then washed with phosphate-buffered saline three times and incubated at 37°C in an atmosphere of 5% CO2. Samples were collected on the day of infection and then daily for a maximum of 10 days. No virus controls were necessary for these cells, since inocula had been removed and the cells were washed before further incubation and sampling.

Plaque assay. Plaque assays for determination of virus titers in cell culture supernatants were performed using Vero cells with an agarose double overlay and neutral red for chimeric viruses and YF 17D virus and by immunocytochemical focus-forming assay for wild-type DEN viruses as previously described.2,24 Titers in PFU/mL were determined for each time point and plotted as a function of time to generate a virus growth curve in each cell type.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth kinetics in DCs. In our preliminary experiments, the growth kinetics of the different viruses in both sources of DCs (NHDCs and iNemod DCs) showed similar growth patterns. We later selected iNemod DCs for further studies (data for iNemod DCs are shown in Figure 1Go). In general, all ChimeriVaxTM-DEN1-4 and wild-type DEN1-4 viruses derived from Vero cells grew to similar titers (3–4 log10 PFU/mL) to YF 17D virus regardless of the DC source. During the first 24 hours of culture, there was a decrease in the concentration of all viruses. By 48 hours post-infection, no infectious virus could be detected in virus control (samples that contained no DCs) samples, whereas virus titers in the presence of DCs reached a peak titer of 2–4.5 log10 PFU/mL and remained above virus control samples through 96 hours of culture (Figure 1Go). Loss of virus potency in virus control samples at 48 hours was probably due to thermal inactivation of the virus (there was no stabilizer in the medium), which is consistent with the stability data for these chimeras at 37°C (Guirakhoo F, unpublished data). Both YF 17D and ChimeriVaxTM-DEN1-4 viruses exhibited similar growth kinetics in DCs. The peak titers (~3–4.5 log10 PFU/mL) for all chimeras and YF 17D virus were reached between 48 and 72 hours post-infection at an MOI of 0.1. The peak titers of ChimeriVax-DEN2 (Figure 1BGo) and ChimeriVaxTM-DEN4 (Figure 1DGo) viruses were slightly higher than that of YF 17D virus, whereas the peak titers of ChimeriVaxTM-DEN1 (Figure 1AGo) and ChimeriVaxTM-DEN3 (Figure 1CGo) viruses were slightly lower than YF 17D virus.



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  		    FIGURE 1. Growth kinetics of ChimeriVaxTM-DEN1-4 viruses versus Vero-derived wild-type (wt) dengue (DEN)1-4 viruses and yellow fever (YF) 17D virus in dendritic cells (DC) (i-NemodDCTM) infected at a multiplicity of infection of 0.1. Cell-free supernatants were collected every day for four days. Virus output was determined by plaque assay. Virus control (VC) was the same virus inoculum used for the DC infection, at the same concentration, incubated in cell culture medium, without cells. The graph represents the results of a plaque assay experiment, performed in duplicate, for each sample collected. A, ChimeriVaxTM-DEN1; B, ChimeriVaxTM-DEN2; C, ChimeriVaxTM-DEN3; D, ChimeriVaxTM-DEN4. PFU = plaque-forming units.

 
Wild-type DEN1-4 derived from 3–4 passages in Vero cells also had similar growth kinetics as the chimeras (also Vero cell derived) in DCs at similar MOIs (e.g., 0.1) with peak titers at 48 hours post-infection. The titers of wild-type DEN1 and wild-type DEN3 viruses were slightly lower than those of YF 17D virus (Figure 1AGo and Figure 1CGo), whereas wild-type DEN2 and wild-type DEN4 viruses were slightly higher (Figure 1B and 1DGo). In contrast, wild-type DEN1-4 viruses grown in C6/36 mosquito cells did not show any growth in DCs, except for wild-type DEN2 virus. This virus (derived from C6/36 cells) grew to a modest peak titer of 2.5 log10 PFU/mL at 48–72 hours in DCs. However, when it was passaged in mammalian (Vero) cells, it grew to 4.5 log10 in DCs infected at a similar MOI to the C6/36-derived virus. The difference was even more apparent for DEN1, 3, and 4. At similar MOIs, C6/36-derived wild-type DEN1,3, and 4 did not show any growth in DCs, whereas the same viruses passaged in Vero cells grew to 3 log10 PFU/mL (wild-type DEN1) and to 4–4.5 log10 PFU/mL (wild-type DEN3 and wild-type DEN4).

Growth kinetics in hepatic cell lines. In Huh7 cells, the differences in growth of YF 17D, ChimeriVaxTM-DEN1-4 and wild-type DEN1-4 viruses were not as dramatic as those of HepG2 or THLE3 cells. ChimeriVaxTM-DEN1, 2, and 4 viruses grew slightly faster and to a higher titer (peak titer of ~8.5 log10 PFU/mL on day 3) than YF 17D virus (peak titer of ~8.0 log10 PFU/mL on day 4) (Figure 2A, B, and DGo), whereas ChimeriVaxTM-DEN3 grew to a peak titer of ~7.4 log10 PFU/mL on day 4 (Figure 2 CGo). Wild-type parent DEN1, 2, and 3 viruses grew slower and to lower titers than the chimeras or YF 17D viruses (peak titers ~7.5 log10 PFU/mL on day 7). Wild-type DEN4 virus grew slower than all other viruses and reached the peak titer of only ~6.9 log10 PFU/mL in Huh7 cells (Figure 2 DGo). The maximum cytopathic effect (CPE) was observed at day 6 for YF 17D and ChimeriVaxTM-DEN1, 3, and 4 viruses (day 5 for ChimeriVaxTM-DEN2).



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  		    FIGURE 2. Growth kinetics of ChimeriVaxTM-DEN1-4 viruses, wild-type (wt) dengue (DEN)1-4 viruses, and yellow fever (YF) 17D virus in Huh7 cells at a multiplicity of infection of 0.001. Cell-free supernatants were collected every day for 10 days or until a complete cytopathic effect was observed. Virus output was determined by plaque assay. A, ChimeriVaxTM-DEN1; B, ChimeriVaxTM-DEN2; C, ChimeriVaxTM-DEN3; D, ChimeriVaxTM-DEN4. PFU = plaque-forming units.

 
In HepG2 cells, YF 17D virus grew to 7.9 log10 by day 3. ChimeriVaxTM-DEN1-4 viruses also peaked on day 3, but their titers remained low (4.3, 2.3, 3.8, and 5.4 log10 PFU/mL for ChimeriVaxTM DEN1 to 4, respectively). Wild-type DEN1-3 viruses grew to 3.8, 3.7, and 3.7 log10 PFU/mL, respectively, on day 6, whereas wild-type DEN4 virus grew to a peak titer of 4.4 log10 PFU/mL on day 3 (Figure 3Go). In HepG2 cells, the peak titers of ChimeriVaxTM-DEN1-4 were between 2 and 5 log10 lower than YF 17D virus, with ChimeriVaxTM-DEN4 showing the highest titer and ChimeriVaxTM-DEN2 showing the lowest. The maximum CPE was observed at day 7 for YF 17D virus, while ChimeriVaxTM-DEN1-4 and wild-type DEN1-4 showed little to no CPE out to day 10.



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  		    FIGURE 3. Growth kinetics of ChimeriVaxTM-DEN1-4 viruses, wild-type (wt) dengue (DEN)1-4 viruses, and yellow fever (YF) 17D virus in HepG2 cells at a multiplicity of infection of 0.001. Cell-free supernatants were collected every day for 10 days or until a complete cytopathic effect was observed. Virus output was determined by plaque assay. A, ChimeriVaxTM-DEN1; B, ChimeriVaxTM-DEN2; C, ChimeriVaxTM-DEN3; D, ChimeriVaxTM-DEN4. PFU = plaque-forming units.

 
In THLE-3 hepatic cells, YF 17D virus grew to a peak titer of 5.6 log10 PFU/mL at four days post-infection (Figure 4Go). ChimeriVaxTM-DEN1-4 viruses showed markedly lower growth, with peak titers between 1 and 5.5 logs lower than YF 17D virus, with ChimeriVaxTM-DEN1 virus showing no growth (1 log10 PFU/mL) and ChimeriVaxTM-DEN4 virus showing the highest titers (3.9 log10 PFU/mL) (Figure 4Go). Wild-type DEN1-4 viruses showed little to no growth in THLE-3 cells. The DEN1, DEN2, and DEN4 wild-type viruses had lower titers than their corresponding ChimeriVaxTM-DEN1-4 vaccines, while DEN3 virus showed higher titers (2.3 log10 PFU/mL on day 4) than ChimeriVaxTM-DEN3 (1.5 log10 PFU/mL on day 4). Due to the nature of this cell line (partially adherent), it was not possible to determine the CPE.



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  		    FIGURE 4. Growth kinetics of ChimeriVaxTM-DEN1-4 viruses, wild-type (wt) dengue (DEN)1-4 viruses, and yellow fever (YF) 17D virus in THLE-3 cells at a multiplicity of infection of 0.001. Cell-free supernatants were collected every day for 10 days or until a complete cytopathic effect was observed. Virus output was determined by plaque assay. A, ChimeriVaxTM-DEN1; B, ChimeriVaxTM-DEN2; C, ChimeriVaxTM-DEN3; D, ChimeriVaxTM-DEN4. PFU = plaque-forming units.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Flaviviruses, like other arthropod-borne viruses, gain access to the body by the bite of blood-feeding mosquitoes or ticks. The sites of deposition of mosquito or tick saliva containing virus are the epidermis and dermis. However, the interactions between virus deposited in this fashion and host cells are not well understood. It was shown that human Langerhans cells might be the initial target for replication of DEN virus, and that intradermal inoculation of non-human primates or mice with DEN virus resulted in local proliferation and active migration of Langerhans cells.5,25,26 Moreover, resident immature DCs of skin were shown to be the cells most permissive for DEN infection, as demonstrated in a skin biopsy from a DEN vaccine recipient.8 Initial experiments demonstrated that blood-derived DCs were 10-fold more permissive for DEN virus infection than monocytes or macrophages.5 Although these studies have demonstrated involvement of DCs in the initiation process of DEN virus, no data have been available for YF 17D virus or any other chimeras that use YF 17D virus as a vector. We investigated the interaction of ChimeriVax-DEN1-4 and blood-derived human DCs in vitro. Surprisingly, we found that these cells were 5–10-fold more efficient in replicating ChimeriVaxTM-DEN2 or -DEN4 vaccine viruses (YF 17D virus with the prM and E genes of DEN2 or DEN4 viruses) than their wild-type DEN2, DEN4, or YF 17D parent viruses. The production of ChimeriVaxTM-DEN1 and DEN3 were similar to their wild-type parent viruses, but slightly lower than YF virus 17D. It is possible that the binding of DEN1 and DEN3 viruses to DCs is less efficient than that of DEN2 and DEN4 chimeras. The level of virus binding to human leukocyte cell lines was shown to vary widely between cell lines, and, notably, between virus strains within a DEN serotype.27 A single mutation from Asn to Asp at amino acid 390 in the E protein of the DEN2 virus (strain New Guinea C) reduced virus output from DCs, indicating that envelope structures can affect replication of DEN viruses in DCs.28 We also found that DCs infected with C6/36 cell-derived viruses produced less virus (by a factor of 1 log10 PFU/mL or greater) than Vero cell-derived viruses infected at similar MOIs. It has been shown that some strains of C6/36-derived wild-type DEN1-4 viruses can infect and replicate efficiently in DCs at higher MOIs than the MOI used in our experiments.5–7,10 When DCs were infected with C6/36-derived wild-type DEN2 virus at a higher MOI (4 instead of 0.4), the level of virus production was consequently increased 10-fold (from 2.7 to 3.7 log10 PFU/mL). This was, however, not true for Vero cell-derived viruses because these viruses efficiently replicated at lower MOIs in DCs. The observed differences concerning the growth kinetics of mosquito and mammalian derived DEN1-4 suggest that the cell type used to produce the virus might be able to alter viral behavior in DCs.

The E glycoproteins of DEN1 and DEN3 viruses are glycosylated at Asn 67 and Asn 153, whereas DEN2 and DEN4 viruses are glycosylated only at Asn 67.29 In mosquito cells, the carbohydrate residues are of high mannose, whereas in mammalian cells these are of a complex type. It has been suggested that the high-mannose N-oligosaccharide at Asn 153 of DEN1 and DEN3 viruses produced in mosquito cells might account for the higher affinity of the E glycoporotein binding to the DC-SIGN-expressing cells.10 Since in our experiments the level of virus output with ChimeriVax-DEN1 and -DEN3 viruses was lower than that of ChimeriVax-DEN2 and -DEN4 viruses, and the fact that infection of DCs with Vero cell-derived viruses resulted in a higher virus output than that of mosquito derived viruses, it is possible that Vero cell-derived viruses may have been independent on DC-SIGN receptors expressed on DCs. Alternative receptors for infection of DCs have also been suggested for West Nile virus, since this virus, which carries a carbohydrate at residue 153, was not dependent on DC-SIGN for replication in DC-SIGN-expressing THP-1 cells.10 Alternatively, it is likely that the attenuated Vero cell-derived DEN viruses induce a different cytokine milieu that modulates the infection of DCs, or that the virulent wt mosquito cell-derived virus shuts down macromolecular synthesis of the cells, induces apoptosis, or has another deleterious effect on viral replication. Because we measured only the production of infectious virus output, we cannot exclude the possibility that the replication of C6/36-and Vero cell-derived viruses in DCs were similar, and that in DCs infected with C6/36-derived viruses progeny particles were not assembled, released, or produced in non-infectious forms (e.g., containing uncleaved PrM proteins). It should also be kept in mind that conclusion about differences or similarities of results reported in different laboratories about tropism of DEN viruses for DCs should be made cautiously, since the source and methods of preparation of DCs, virus strains, cell culture passage history of viruses, MOI of infection, and other variable factors may have influenced the outcome. It was shown that a DEN2 strain (southeast Asian genotype, 16681) that replicates more efficiently in DCs is also associated with increased virulence in humans.28 It is unlikely that a slightly higher replication of YF-DEN2 and YF-DEN4 chimeras, compared with their parent wt viruses, in DCs, may indicate that these viruses are equally or more virulent than their parent viruses because 1) the magnitude of viremia in ChimeriVax-DEN1-4 immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wt DEN viruses,2,23,30,31 and 2) ChimeriVax-DEN2 virus induced a low level of transient viremia and high level of neutralizing antibodies, which lasted more than a year upon a single immunization of humans (Kitchener S, unpublished data). Since this vaccine was well tolerated without causing any dengue-like illness or serious AEs, a slightly higher than wild-type replication in DCs will not indicate that ChimeriVax-DEN2 is equally or more virulent than wild-type DEN viruses. In any case, the ChimeriVaxTM vaccines, which are grown in Vero cells, would appear to be ideally suited for presentation to and replication in DCs, would not impair DC function, and would be expected to induce a robust immune response in the host if presented preferentially to these cells.

The replication of chimeric vaccines in DCs also suggests that an epidermal or intradermal administration of ChimeriVaxTM-DEN1-4 vaccine may be a more efficient method of immunization than the subcutaneous route, by directly targeting Langerhans cells. Studies comparing inactivated West Nile virus to live virus showed that only live virus was able to induce maturation and migration of Langerhans cells to local lymph nodes and to generate antibody responses and cytotoxic T lymphocytes.11

In contrast to the study in DCs, which evaluated infectivity and virus production, the experiments in hepatic cell lines tested the pathogenicity of the viruses. Clinical studies have demonstrated that both DEN and YF 17D viruses can cause hepatic damage in susceptible persons.21,22 Infection of hepatic cell lines with chimeric YF-DEN and their parent viruses, wild-type DEN1-4 and YF 17D, showed a significant difference in the growth kinetics of YF 17D virus and ChimeriVaxTM-DEN1-4 viruses in both HepG2 and THLE-3 cells. The YF 17D virus produced higher titers and caused an extensive CPE earlier than ChimeriVaxTM-DEN1-4 or wild-type DEN1-4 viruses. This data was consistent with another study, which compared the growth kinetics of wild-type YF virus to wild-type DEN1 virus in HepG2 cells.32 In their experiments, the titer of wild-type DEN1 virus (strain Oster) was similar to the titer we observed with wild-type DEN1 virus (strain PUO359) (~3 log10 PFU/mL), which was 3–5 log10 PFU/mL lower than YF virus. In addition, ultrastructural analyses showed that DEN virus-infected HepG2 cells had fewer virus particles in vesicles and in the cytoplasm. It is possible that early apoptosis of hepatocytes leads to limited foci of infection and low production of viral progeny in both HepG2 and THLE-3 cells infected with ChimeriVaxTM-DEN or wild-type DEN viruses. To our knowledge, there is no published report on the level of YF 17D virus replication in human hepatic cells in vitro; however, extensive pathologic changes in the liver have been demonstrated in cases of YF 17D virus vaccine failure, as well as in chick embryos that died during the production of YF 17D virus vaccine.22,33

Of the three cell lines tested, THLE-3 may more closely mimic normal human liver cells than either HepG2 or Huh7 because THLE-3 is derived from normal liver cells that are immortalized via SV40 T antigen, whereas HepG2 and Huh7 are carcinoma derived. ChimeriVaxTM-DEN1-4 replicated to significantly lower titers than YF-VAX® in both HepG2 and THLE-3 cells, but not in Huh7 cells. Many investigators have shown that Huh7 cells (grown in tissue culture) are permissive to replication of many viruses, irrespective of their attenuated phenotype. Moreover, replication of chimeric DEN viruses in these cells may be similar to those of Vero cells, which are used to manufacture vaccine viruses. It seems that the Huh7 cells may be more appropriate for other applications, such as titration of viruses (e.g., in a plaque assay, where certain viruses produce small plaque which are not easy to read) rather than prediction of hepatotropism of viruses. However, Huh7 cells were shown to be useful in differentiating attenuated and virulent DEN viruses (in vivo) when transplanted into severe combined immunodeficient (SCID) mice.34 Because the relevance of the Huh7-SCID model to the pathogenesis of flavivirus infections in humans is uncertain, and the fact that extrapolating in vitro results to the situation in vivo is difficult, we are currently performing studies to define the sites of replication of chimeras and YF 17D virus in vivo in relevant animal models. In any case, replication of all chimeras in HepG2 and THLE 3 cells was dramatically lower (~2–5 logs) than that of YF-VAX, which has been used in more than 400 million people over a period of > 60 years with only few cases of hepatic failure in susceptible individuals.20 Chimeric YF-Japanese encephalitis, YF-West Nile, and YF-DEN viruses have been used in multiple studies in non-human primates (toxicology tests) and human studies without any sign of liver toxicity. As we mentioned earlier, the lower growth of wild-type DEN viruses in hepatic cells may be due to an apoptosis phenomena, which have been shown with wild-type DEN viruses in liver cells.

In summary, it is possible that the ChimeriVaxTM-DEN1-4 may generate a strong immune response similar to YF 17D virus, but with less liver toxicity. This study also shows that there are distinct differences in the ability of DEN viruses derived from mosquito or mammalian cells to replicate in DCs. Therefore, the cell line used to produce the vaccines may significantly affect their replication in human skin DCs and induction of an immune response. The permissiveness of DCs for ChimeriVaxTM-DEN1-4 supports the idea of an epidermal vaccination as an efficient route of delivery for these viruses. It is possible that targeting epidermal DCs might enhance replication of the vaccine viruses in vivo. The safety and tolerability of the chimeric vaccines delivered by epidermal immunization of humans remain to be determined in clinical trials.

Received March 16, 2004. Accepted for publication August 6, 2004.

Acknowledgments: We thank P. Papastathis (Acambis, Inc.) for excellent technical assistance and cell culture support.

Financial support: This work was supported by Aventis Pasteur (Marcy-L’Etoile, France).

Disclosure: Farshad Guirakhoo holds stock of Acambis, Inc. and is currently conducting research sponsored by this company. This statement is made in the interest of full disclosure and not because the author considers this to be a conflict of interest.

Authors’ addresses: Samantha Brandler, Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Nathan Brown, Thomas H. Ermak, Fred Mitchell, Megan Parsons, Zhenxi Zhang, Thomas P. Monath, and Farshad Guirakhoo, Acambis, Inc., 38 Sidney Street, Cambridge, MA 02139, Telephone: 617–761–4323, Fax: 617–494–1741, E-mail: farshad.guirakhoo{at}acambis.com. Jean Lang, Aventis Pasteur, Campus Merieux, 1541 Avenue Marcel Merieux, Marcy-L’Etoile F-69280, France.

Reprint requests: Farshad Guirakhoo, Acambis, Inc., 38 Sidney Street, Cambridge, MA 02139.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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