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

Three dengue virus type 4 (DENV-4) vaccine candidates containing deletions in the 3′ noncoding region were prepared by passage in DBS-FRhL-2 (FRhL) cells. Unexpectedly, these vaccine candidates and parental DENV-4 similarly passaged in the same cells failed to elicit either viremia or a virus-neutralizing antibody response. Consensus sequence analysis revealed that each of the three viruses, as well as the parental DENV-4 when passaged in FRhL cells, rapidly acquired a single Glu₃₂₇-Gly substitution in domain III (DIII) of the envelope protein (E). These variants appear to have accumulated in response to growth adaptation to FRhL cells as shown by growth analysis, and the mutation was not detected in the virus following passage in C6/36 cells, primary African green monkey kidney cells, or Vero cells. The Glu₃₂₇-Gly substitution was predicted by molecular modeling to increase the net positive charge on the surface of E. The Glu₃₂₇-Gly variant of the full-length DENV-4 selected after three passages in FRhL cells showed increased affinity for heparan sulfate compared to the unpassaged DENV-4, as measured by heparin binding and infectivity inhibition assays. Evidence indicates that the Glu₃₂₇-Gly mutation in DIII of the DENV-4 E protein was responsible for reduced infectivity and immunogenicity in rhesus monkeys. Our results point out the importance of cell substrates for vaccine preparation since the virus may change during passages in certain cells through adaptive selection, and such mutations may affect cell tropism, virulence, and vaccine efficacy.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 tick-borne 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 ∼9-kDa membrane protein (M) and a larger ∼54-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 for coordination of various cellular functions (10), the role of HS in mediating viral entry for infection of susceptible mammalian hosts or insect vectors is less defined. Studies have shown that propagation of wild-type strains of DENV adaptively selects variants to replicate in certain mammalian cell cultures, including rodent-derived BHK-21 (kidney fibroblast) and human SW-13 (adrenal carcinoma) cell lines (28, 33). These variants acquire mutations in E, creating new HS binding sites and facilitating interactions to gain an entry into these cells. Such adaptive selection of variants involving binding to HS also appears to be a common mechanism for other single-stranded RNA viruses, including alphaviruses and foot-and-mouth disease virus. Analysis of the biological properties of these HS binding variants demonstrated attenuation of virulence and restriction of cell tropism (2, 4, 15, 18, 26, 49).Passage of wild-type YFV in animals and in cell cultures was successfully employed to produce the live-attenuated 17D vaccine several decades ago, and, more recently, the live-attenuated Japanese encephalitis vaccine SA14-14-2 was similarly produced (17, 39). In an effort to develop a DENV vaccine, investigators have sought to attenuate the virus by serial passage in primary dog kidney (PDK) cells or selection of small plaque-forming viruses in cultured cells (9, 14). Depending on the DENV serotype, various passage levels in PDK cells have been empirically derived in order to produce attenuated live vaccines. Final passage in fetal rhesus lung (FRhL) cells is frequently used for virus seed and vaccine lot production (8). The FRhL cell strain is a normal diploid cell suitable for the production of vaccines for human use (55). These cells have been shown to support the replication of all four DENV serotypes to high titers (9, 31).The availability of DENV cDNA clones has made it possible to modify the viral genome in order to derive growth-restricted and chimeric DENV mutants for the production of live vaccine candidates (7, 22, 23). Earlier, a series of DENV-4 mutants containing deletions in the 3′ noncoding region (NCR) was generated and shown to be attenuated for replication in cultured cells and in nonhuman primates (36). In an effort to develop DENV vaccine candidates with defined attenuating mutations, these viruses were propagated in FRhL cells for vaccine production and then tested in rhesus macaques. Unexpectedly, the animals failed to develop either antibody responses or viremia after inoculation. In the present report, we present evidence that passage of these DENV-4 constructs in FRhL cells rapidly selected for variants containing a single mutation in E that resulted in increased virus binding to heparin, a highly sulfated form of HS, and the loss of infectivity for primates.