Abstract. The recombinant dengue virus type-4 vaccine candidate 2A⌬30 was attenuated in rhesus monkeys due to an engineered 30-nucleotide deletion in the 3Ј-untranslated region of the viral genome. A clinical trial to evaluate the safety and immunogenicity of a single dose of 2A⌬30 was conducted with 20 adult human volunteers. The vaccine candidate was well tolerated and did not cause systemic illness in any of the 20 volunteers. Viremia was detectable in 14 volunteers at a mean level of 1.6 log 10 plaque-forming units/ml of serum, although all 20 volunteers seroconverted with a seven-fold or greater increase in serum neutralizing antibody titer on day 28 post-vaccination (mean titer ϭ 1:580). A mild, asymptomatic, macular rash developed in 10 volunteers, and a transient elevation in the serum level of alanine aminotransferase was noted in five volunteers. The low level of reactogenicity and high degree of immunogenicity of this vaccine candidate warrant its further evaluation and its use to create chimeric vaccine viruses expressing the structural genes of dengue virus types 1, 2, and 3.

Langat virus (LGT) strain TP21 is the most attenuated of the tick-borne flaviviruses for humans. Even though LGT has low-level neurovirulence for humans, it, and its more attenuated egg-passage derivative, strain E5, exhibit significant neurovirulence and neuroinvasiveness in normal mice, albeit less than that associated with tick-borne encephalitis virus (TBEV), the most virulent of the tick-borne flaviviruses. We sought to reduce or ablate these viral phenotypes of TP21 and E5 by using a strategy that had been used successfully in the past to reduce neurovirulence and abolish neuroinvasiveness of TBEV, namely substitution of structural protein genes of the tick-borne flavivirus for the corresponding genes of dengue type 4 virus (DEN4). In pursuit of these objectives different combinations of LGT genes were substituted into the DEN4 genome but only chimeras containing LGT structural proteins premembrane (preM) and envelope glycoprotein (E) were viable. The infectious LGT(preM-E)͞DEN4 chimeras were restricted in replication in simian cell cultures but grew to moderately high titer in mosquito cell culture. Also, the chimeras were at least 5,000 times less neurovirulent than their parental LGT virus in suckling mice. Significantly, the chimeras lacked detectable evidence of neuroinvasiveness after i.p. inoculation of Swiss mice or the more permissive SCID mice with 10 5 or 10 7 plaque-forming units (PFU), respectively. Nonetheless, i.p. inoculation of Swiss mice with 10 or 10 3 PFU of either chimeric virus induced LGT neutralizing antibodies and resistance to fatal encephalitis caused by i.p. challenge with LGT TP21. The implications of these observations for development of a live attenuated TBEV vaccine are discussed.Tick-borne flaviviruses are endemic throughout most of the Northern Hemisphere, causing disease of varying severity that can have a mortality as high as 20-30% (1). Similar to all flaviviruses, viruses of the tick-borne group have a positive sense nonsegmented RNA genome that encodes a single long polyprotein that is processed to yield capsid (C), premembrane (preM), envelope glycoprotein (E) structural proteins followed by nonstructural protein NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 in that order (2, 3). These tick-borne viruses share envelope glycoprotein epitopes that often induce cross-resistance among viruses of the group. These properties of antigenic crossreactivity and virulence polymorphism suggested that successful immunization might be achieved by using a live, naturally attenuated tick-borne flavivirus. The impetus for this approach was the recovery of a virus from ticks in Malaysia, namely Langat virus (LGT), that did not appear to be associated with human disease under natural conditions (4, 5).Approximately 30 years ago Yelantsev virus (6), which subsequently was shown to be identical to wild-type LGT, strain TP21 (7), was evaluated in 649,479 individuals as a candidate live attenuated vaccine for prevention of tick-borne encephalitis. Studies were discontinued when it was ...

The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3 noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. Immediately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK 2 cells compared with the full-length RNA transcripts of wild-type parental virus. Deletion mutations that extended as far as the 5 boundary of the 3 noncoding region and whose 3 boundary did not extend beyond the last 113 nt of the 3 end were viable. With the exception of mutant 3d 303-183, which contained a deletion of nt 303 to 183 from the 3 terminus, deletion mutants produced plaques that appeared late on simian LLC-MK 2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK 2 cells than parental wild-type virus. Significantly, mutant 3d 303-183 grew to a high titer and was least restricted in growth. Mutant 3d 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys. There was a suggestion that occurrence and duration of viremia were reduced for some of the deletion mutants compared with the wild-type virus. However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection. Mutant 3d 303-183 induced an antibody response equivalent to that stimulated by wild-type virus, whereas other mutants induced low to moderate levels of antibodies, as measured by radioimmunoprecipitation and virus neutralization. The immunogenicity of these 3 DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK 2 cells. One or more mutants described in this paper may prove to be useful for immunization of humans against disease caused by dengue virus.

The antigenic site of dengue type 2 virus (DEN2)-neutralizing monoclonal antibody (mab) 3H5 was investigated by mutational analysis. Sequence comparisons indicated that much of the 12-amino-acid sequence extending from position 386 to 397 of the DEN2 envelope glycoprotein (E) previously thought to represent the DEN2-specific mab 3H5 binding site was also present in some dengue type 1, 3, or 4 virus strains. However, the region occupied by the Glu-Pro-Gly sequence at upstream positions 383 to 385 was completely conserved among DEN2 strains, but divergent in other serotype viruses, suggesting that this sequence might be part of the antigenic site of mab 3H5. We investigated this possibility by employing the previously constructed chimeric DEN2(PreM-E)/DEN4 cDNA clone to produce viable mutants bearing DEN2 PreM and E sequences that could be analyzed for binding to and neutralization by mab 3H5. We constructed 13 such DEN2 mutants that contained a single amino acid substitution in the region between positions 383 and 393 of DEN2 E. Each single substitution in the region spanning positions 386 through 393 of DEN2 yielded a virus that was as reactive with mab 3H5 as the parental chimeric virus. These results are consistent with the extent of sequence conservation in the region. In contrast, 5 of 6 mutants that sustained an amino acid substitution at position 383, 384, or 385 failed to react with mab 3H5 as detected by immunofluorescence assay and failed to be neutralized by the mab. Interestingly, each of the 5 mab-resistant DEN2 mutants also exhibited reduced mouse neurovirulence compared to parental chimeric DEN2 when inoculated intracerebrally. These observations suggest that the Glu-Pro-Gly sequence at positions 383-386 of the DEN2 E is a component of the site against which mab 3H5 is directed. In the recently determined three-dimensional structure of the related tick-borne encephalitis virus E, the Glu-Pro-Gly sequence would be located on the lateral surface of the immunoglobulin-like domain that is proposed to bind to the host cell receptor.

A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V H and V L sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V H sequences but varied in their V L sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Among the arthropod-borne flaviviruses, the four dengue virus serotypes (dengue type 1 virus [DENV-1], DENV-2, DENV-3, and DENV-4) that constitute a serologically distinct subgroup are most important in terms of human morbidity and geographic distribution. Dengue viruses cause dengue outbreaks and major epidemics in most tropical and subtropical areas where Aedes albopictus and Aedes aegypti mosquitos are abundant. Dengue virus infection produces fever, rash, and joint pain in humans. A more severe and life-threatening form of dengue, characterized by hemorrhagic fever and hemorrhagic shock, has occurred with increasing frequency in Southeast Asia and Central and South America, where all four dengue virus serotypes circulate. The underlying cause of severe dengue remains controversial (23,53). An association of severe dengue with increased viral replication has been reported recently (61). A safe and effective vaccine against dengue is currently not available.The dengue virus contains a positive-strand RNA genome coding for a polyprotein that is cleaved co-and posttranslationally by a combination of cellular and viral proteases to generate the individual viral proteins (9,19,40). Dengue virus prM and E structural proteins and nonstructural NS1 protein are glycosylated. The prM glycoprotein is further cleaved by the cellular enzyme furin following viral assembly, generating M, which is present in the mature virus (58). Flavivirus prM and E form heterodimers, which are assembled into viral particles during infection (62). In this manner, the prM serves to protect the functional integrity of E from acid-induced conformational change (26,31). The E glycoprotein is responsible for cell attachment, possibly mediated by a receptor, and for fusion with the cell membranes following viral entry.Mouse monoclonal antibodies against the dengue viruses have been valuable for dengue virus serotype determination (20,27). Studies in which monoclonal antibodies were used against dengue virus and other flaviviruses have also provided valuable...

The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys 174 -Glu substitution and another contained a Pro 176 -Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18-to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 ⌬D, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 ⌬D at 20 g/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 ⌬D were completely protected against 100 50% monkey infectious doses (MID 50 ) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys 174 -Glu substitution identical to that found in vitro was isolated from monkeys challenged with 10 6 MID 50 of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.The four dengue virus serotypes (dengue virus types 1 to 4 [DENV-1 to DENV-4]) cause more morbidity in humans than any other arthropod-borne flaviviruses (40). Up to 100 million DENV infections occur every year, mostly in tropical and subtropical areas where the vector mosquitoes, principally Aedes aegypti and Aedes albopictus, are present. Dengue illnesses range from mild fever to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which has fatality rates ranging from Ͻ1% to 5% in children. The most severe dengue (Ͼ90% fatality rate) occurs in patients reinfected with DENV of a serotype different from that in the primary infection (15, 50). Antibody-dependent enhancement (ADE) of DENV replication has been proposed as an underlying pathogenic mechanism of severe DHF/DSS (17). A safe and effective vaccine against dengue is still not available.Early studies of DENV infections in human volunteers showed that homotypic immunity against the same serotype is life-long but that heterotypic immunity against other serotypes lasts only months (49). Since antibodies provide the important component of acquired immunity against infection, type-specific immunity afforded by antibody may contribute significantly to long-term protection. Antigenic differences exist among strains of the same serotype (21). DENV variants that form ...

No 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 ...