Passage of Dengue Virus Type 4 Vaccine Candidates in Fetal Rhesus Lung Cells Selects Heparin-Sensitive Variants That Result in Loss of Infectivity and Immunogenicity in Rhesus Macaques

Abstract

Dengue virus (DENV) infects humans via the bite of infected mosquitoes, principally Aedes aegypti. DENV infections can be asymptomatic or cause a spectrum of illnesses that range from mild dengue fever to a severe, life-threatening disease characterized by dengue hemorrhagic fever/dengue shock syndrome (13, 38). The four DENV serotypes (DENV type 1 [DENV-1] to DENV-4) are the most important members of the genus Flavivirus in terms of morbidity, geographic distribution, and socioeconomic burden (1, 12). Several other members of the flaviviruses, including yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus, and tickborne encephalitis virus, are also important human pathogens.The flavivirus virion is a spherical enveloped particle with icosahedral symmetry. It has a relatively simple structure, consisting of an inner nucleocapsid-virus RNA core and an outer lipid bilayer membrane into which a small ϳ9-kDa membrane protein (M) and a larger ϳ54-kDa envelope protein (E) are embedded. The E protein, which is approximately 500 amino acids in length, is the major antigen responsible for attachment to the cell surface, viral entry mediated by endocytosis, fusion with endosomal membranes, and the eliciting of host immune responses. There are 180 copies of E in the form of homodimers arranged in a tight array on the smooth virion surface without major spikes (21,37,48). Structural analysis indicates that each E monomer is folded into three structurally distinct domains, termed domains I, II, and III (DI, DII, and DIII, respectively). DIII has an immunoglobulin-like fold, a structural feature shared by many cell-adhesive molecules and receptor-binding proteins. DIII has been proposed to be responsible for binding interaction with cell surface receptors (16,48). A number of mosquito-borne flavivirus E proteins contain a sequence motif in DIII that is recognized by integrin receptors. Mutations affecting cell attachment that cluster in this region are associated with attenuation of virulence and cell tropism (26,27,29,43,53).A specific cell surface receptor has not been clearly identified for DENV or any other flavivirus. Studies focusing on the mechanisms of viral binding and entry in mosquito C6/36 cells (42, 56) or mammalian cells (35,41) have suggested a number of proteins of various sizes that are capable of binding the DENV virion. Recently, the C-type lectin DC-SIGN was found to be capable of facilitating DENV infection of dendritic cells (51,52). It has been proposed that flaviviruses could also utilize other less specific molecules on the cell surface as coreceptors for initial adsorption and infection. Infection of DENV-2 was first found to depend on heparan sulfate (HS), a major constituent of the extracellular matrix and a surface component of most mammalian cells, for binding interaction and infectivity of cultured cells (6). In that study the authors identified sequences of two HS binding sites in E, one in DIII and the other in the junction between DI and DIII. Although HS is essential ...

Resistance to Dengue Virus Infection in Mice Is Potentiated by CXCL10 and Is Independent of CXCL10-Mediated Leukocyte Recruitment

“…These data clearly demonstrated that the reduction of DENV binding to heparan sulfate in neuronal cells decreases DENV infectivity in neuronal cells. In contrast to the results of our study, two reports demonstrated the reduction of DENV binding to heparan sulfate leads to the increase of DENV infectivity in hosts (47,48). Añez et al (48) reported that s.c. infection of primates with a DENV-4 mutant that has high binding affinity for heparin resulted in a reduced infectivity; similarly, Prestwood et al (47) showed that mice infected i.v.…”

Dengue Type Four Viruses with E-Glu345Lys Adaptive Mutation from MRC-5 Cells Induce Low Viremia but Elicit Potent Neutralizing Antibodies in Rhesus Monkeys

“…The protection against live virus challenges also correlates with anamnestic neutralizing antibodies detected on days 15 and 30 post-challenges. Taken together, the present study demonstrated that the E-E 345 K mutation from MRC-5 cells can still induce potent immunogenicity in rhesus monkeys as compared to the complete loss of monkey immunogenicity by the E-E 327 G mutation reported from FRhL cell passage [11]. These results suggest that the DENV-4 E-E 345 K mutant propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.…”

“…Similar to other flaviviruses, DENV undergo adaptive mutations involving heparan sulfate binding for growth selection in FRhL, BHK and Vero cells [11], [15], [16]…”

Near-Atomic Resolution Cryo-Electron Microscopic Structure of Dengue Serotype 4 Virus

“…The surface potential on DENV2 showed larger continuous patches of positive charges compared to those for DENV1 and DENV4, and this may facilitate enhanced binding to heparan sulfate. It has been shown previously that passaging virus in certain cell lines (FRhL, SW13, and BHK21) may increase its affinity for heparan sulfate (32,33). If this is the case, then the comparison between DENV2 and DENV4 with different passage histories might not be valid.…”