antibody-dependent enhancement ͉ nonhuman primate model ͉ Fc mutations ͉ cross-reactive mAb T he four dengue virus (DENV) serotypes (DENV-1 to DENV-4) are the most important arthropod-borne flaviviruses in terms of morbidity and geographic distribution. Up to 100 million DENV infections occur every year, mostly in tropical and subtropical areas where vector mosquitoes are abundant (1). Infection with any of the DENV serotypes may be asymptomatic or may lead to classic dengue fever or more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which are increasingly common in the dengue endemic areas. Immunity to the same virus serotype (homotypic immunity) is life-long, whereas immunity to different serotypes (heterotypic immunity) lasts 2-3 months so that infection with a different serotype virus is possible (2). DHF/DSS often occurs in patients with second, heterotypic DENV infections or in infants with maternally transferred dengue immunity (3, 4). Severe dengue is a major cause of hospitalization, and fatality rates vary from Ͻ1% to 5% in children.Antibody-dependent enhancement (ADE) has been proposed as an underlying pathogenic mechanism of DHF/DSS (3). ADE occurs because preexisting subneutralizing antibodies and the infecting DENV form complexes that bind to Fc receptorbearing cells, leading to increased virus uptake and replication (4). ADE has been repeatedly demonstrated in vitro using dengue immune sera or monoclonal antibodies and cells of monocytic and recently, B lymphocytic lineages bearing Fc receptors (5-7). ADE of DENV-2 infection has also been demonstrated in monkeys infused with a human dengue immune serum (8).Infection with DENV or any other flavivirus induces broadly cross-reactive but weak or nonneutralizing antibodies (9, 10). These antibodies remain detectable for a long period and rise rapidly during a subsequent heterotypic infection as a result of an anamnestic response. A major subset of these cross-reactive antibodies is directed to immuno-dominant epitopes involving determinants mapped to the flavivirus-conserved fusion peptide in the envelope glycoprotein (E) (11-13). The functional activities of these cross-reactive antibodies are not well characterized.We have identified chimpanzee-human chimeric IgG1 mAbs capable of neutralizing or binding to one or more DENV serotypes (14, 15). Cross-reactive IgG 1A5 neutralizes DENV-1 and DENV-2 more efficiently than DENV-3 and DENV-4, and type-specific IgG 5H2 neutralizes DENV-4 at a high titer (14,15). Analysis of antigenic variants has localized the IgG 1A5 binding site to the conserved fusion peptide in E (11). Thus, IgG 1A5 shares many characteristics with the cross-reactive antibodies detected in flavivirus infections.We investigated the ability of IgG 1A5 to mediate enhancement of DENV replication in monocyte-derived cell lines and in juvenile rhesus monkeys after passive transfer. We also explored strategies to reduce ADE by mutational analysis of the key structures in the Fc of IgG 1A5. A 9-aa deletion at the N termin...
A vectored mucosal vaccine expressing the SARS-coronavirus S protein alone may be highly effective in a single-dose format for the prevention of SARS.
SARS coronavirus (SARS-CoV) administered intranasally and intratracheally to rhesus, cynomolgus and African Green monkeys (AGM) replicated in the respiratory tract but did not induce illness. The titer of serum neutralizing antibodies correlated with the level of virus replication in the respiratory tract (AGM>cynomolgus>rhesus). Moderate to high titers of SARS-CoV with associated interstitial pneumonitis were detected in the lungs of AGMs on day 2 and were resolving by day 4 post-infection. Following challenge of AGMs 2 months later, virus replication was highly restricted and there was no evidence of enhanced disease. These species will be useful for the evaluation of the immunogenicity of candidate vaccines, but the lack of apparent clinical illness in all three species, variability from animal to animal in level of viral replication, and rapid clearance of virus and pneumonitis in AGMs must be taken into account by investigators considering the use of these species in efficacy and challenge studies.
The role of the hepatitis C virus (HCV) p7 protein in the virus life cycle is not known. Previous in vitro data indicated that this 63-aa polypeptide is located in the endoplasmic reticulum and has two transmembrane domains (TMDs) connected by a cytoplasmic loop; the aminoand carboxyl-terminal tails are oriented toward the endoplasmic reticulum lumen. Furthermore, recent in vitro studies suggested that HCV p7 could function as a virus-encoded ion channel. It might therefore be a relevant target for future drug development. We studied the role of HCV p7 in vivo. Because HCV does not replicate efficiently in cell culture, we mutagenized p7 of an infectious genotype 1a cDNA clone and tested RNA transcripts of each mutant for infectivity in chimpanzees by intrahepatic transfection. Appropriate processing of mutant polypeptides was confirmed by studies in transfected mammalian cells. Mutants with deletions of all or part of p7 and a mutant with substitutions of two conserved residues in the cytoplasmic loop were not viable. Thus, p7 is essential for infectivity of HCV. A chimera in which the p7 of the 1a clone was replaced with p7 from an infectious genotype 2a clone also was not viable. This finding suggests a genotype-specific interaction between p7 and other genomic regions. To define which portions of p7 played the most significant role for this interaction, we tested three chimeras with the 1a backbone in which only specific domains of p7 had the 2a sequence. A p7 chimera with 2a tails and TMDs and the 1a cytoplasmic loop was not viable. A mutant with 2a tails and cytoplasmic loop and 1a TMDs also was not viable. However, a p7 chimera with 2a TMDs and cytoplasmic loop and 1a tails was viable. The transfected chimpanzee became viremic at week 2, and recovered viruses had the chimeric sequence. These data indicate that the amino-and͞or carboxyl-terminal intraluminal tails of p7 contain sequences with genotype-specific function. H epatitis C virus (HCV), a member of the Flaviviridae family, is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. HCV is an enveloped virus with a positive single-stranded RNA genome that encodes a single polyprotein precursor of Ϸ3,000 aa that is cleaved into 10 proteins by host or viral proteases (1, 2). These include three structural proteins [one core (C) and two envelope (E1 and E2) glycoproteins], as well as six nonstructural proteins (NS2-NS5B) involved with polyprotein processing and RNA replication. However, it is not known whether the 10th protein, p7, is a structural or nonstructural protein. Studies of HCV subgenomic replicons showed that p7 is not critical for RNA replication (3), but its actual role in the virus life cycle has not been determined. The corresponding protein of bovine viral diarrhea virus (BVDV), another member of the Flaviviridae family, was critical for cell culture infectivity (4). Transfection with RNA transcripts from an infectious BVDV cDNA clone with in-frame deletions or point mutations of p7 could not produce...
The development of a subgenomic replicon derived from the hepatitis C virus (HCV) strain Con1 enabled the study of viral RNA replication in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, increased significantly by a combination of two adaptive mutations in NS3 (E1202G and T1280I) and a single mutation in NS5A (S2197P). However, these cell culture-adaptive mutations influenced in vivo infectivity. After intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, this mutation was detected only in virus genomes recovered from serum at day 4; viruses recovered at day 7 had a reversion back to the original Con1 sequence. Our study demonstrates that mutations that are adaptive for replication of HCV in cell culture may be highly attenuating in vivo. Hepatitis C virus (HCV) is an enveloped virus with a positivestrand RNA genome (1). Worldwide, more than 100 million people are persistently infected with HCV (2). Infected individuals are at increased risk of developing liver cirrhosis and hepatocellular carcinoma. In a subset of infected individuals it is possible to eliminate the infection by therapy with IFN and ribavirin (3,4). Yet, for the development of vaccines or new therapeutics for HCV, it is of great importance to develop cell culture systems to replicate and propagate HCV. The recent construction of a subgenomic replicon, capable of replication in Huh-7 cells, might represent a first step in achieving this goal (5).The HCV genome (Ϸ9,600 nucleotides) has one ORF, which is flanked by UTRs. Translation produces a polyprotein of Ϸ3,000 amino acids that is cleaved into structural [core (C), viral envelope glycoproteins (E1 and E2)], p7, and nonstructural (NS2-5B) proteins by host or viral proteases (6, 7). The NS2 protein, with the N-terminal third of the NS3 protein, is a protease that mediates NS2͞3 cleavage. The N-terminal part of the NS3 protein, with NS4A as a cofactor, has serine-protease activity and mediates the NS3͞4A, 4A͞4B, 4B͞5A, and 5A͞5B cleavages. The carboxy-terminal part of the NS3 protein contains an NTPase and an RNA-helicase. NS4B is a hydrophobic protein that induces the formation of a cytoplasmic membranous structure where all viral proteins are located and that most likely represents the site of RNA replication (8). The NS5A is a phosphorylated protein believed to be important for viral replication. A short region of the NS5A protein is thought to modulate the host IFN-mediated antiviral response (9). Mutations in this region, the IFN sensitivity-determining region, seems to be associated with the sensitivity of HCV genotype 1b viruses to IFN treatment. The protein ...
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