A single amino acid substitution in the envelope protein of chimeric yellow fever-dengue 1 vaccine virus reduces neurovirulence for suckling mice and viremia/viscerotropism for monkeys.

A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78:4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.

Safety and efficacy of chimeric yellow Fever-dengue virus tetravalent vaccine formulations in nonhuman primates.

To construct chimeric YF/DEN viruses (ChimeriVax-DEN), the premembrane (prM) and envelope (E) genes of yellow fever (YF) 17D virus were replaced with those of each wild-type (WT) dengue (DEN) virus representing serotypes 1 to 4. ChimeriVax-DEN1-4 vaccine viruses were prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA (F. Guirakhoo, J. Arroyo, K. V. Pugachev, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratteree, and T. P. Monath, J. Virol. 75:7290-7304, 2001; F. Guirakhoo, K. Pugachev, J. Arroyo, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath, Virology 298:146-159, 2002). Progeny viruses were subjected to three rounds of plaque purifications to produce the Pre-Master Seed viruses at passage 7 (P7). Three further passages were carried out using U.S. current Good Manufacturing Practices (cGMP) to produce the Vaccine Lot (P10) viruses. Preclinical studies demonstrated that the vaccine candidates are replication competent and genetically stable and do not become more neurovirulent upon 20 passages in Vero cells. The safety of a tetravalent vaccine was determined and compared to that of YF-VAX in a formal monkey neurovirulence test. Brain lesions produced by the tetravalent ChimeriVax-DEN vaccine were significantly less severe than those observed with YF-VAX. The immunogenicity and protective efficacy of four different tetravalent formulations were evaluated in cynomolgus monkeys following a single-dose subcutaneous vaccination followed by a virulent virus challenge 6 months later. All monkeys developed low levels of viremia postimmunization, and all the monkeys that had received equal concentrations of either a high-dose (5,5,5,5) or a low-dose (3,3,3,3) formulation seroconverted against all four DEN virus serotypes. Twenty-two (92%) of 24 monkeys were protected as determined by lack of viremia post-challenge. This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.

Viremia and immunogenicity in nonhuman primates of a tetravalent yellow fever-dengue chimeric vaccine: genetic reconstructions, dose adjustment, and antibody responses against wild-type dengue virus isolates.

Chimeric yellow fever (YF)-dengue (DEN) viruses (ChimeriVax-DEN) were reconstructed to correct amino acid substitutions within the envelope genes of original constructs described by Guirakhoo et al. (2001, J. Virol. 75, 7290-7304). Viruses were analyzed and compared to the previous constructs containing mutations in terms of their growth kinetics in Vero cells, neurovirulence in mice, and immunogenicity in monkeys as monovalent or tetravalent formulations. All chimeras grew to high titers [ approximately 7 to 8 log(10), plaque-forming units (PFU)/ml] in Vero cells and were less neurovirulent than YF 17D vaccine in mice. For monkey experiments, the dose of DEN2 chimera was lowered to 3 log(10) PFU in the tetravalent mixture in an effort to reduce its dominant immunogenicity. The magnitude of viremia in ChimeriVax-DEN immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wild-type DEN viruses. All monkeys developed high levels of neutralizing antibodies against homologous (chimeras) or heterologous (wild-type DEN viruses isolated from different geographical regions) viruses after a single dose of monovalent or tetravalent vaccine. Administration of a second dose of tetravalent vaccine 2 months later increased titers to both homologous and heterologous viruses. A dose adjustment for dengue 2 chimera resulted in a more balanced response against dengue 1, 2, and 3 viruses, but a somewhat higher response against chimeric dengue 4 virus. This indicates that further formulations for dose adjustments need to be tested in monkeys to identify an optimal formulation for humans.

Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine.

We previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477-5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ~7.5 log(10) PFU/ml in Vero cells, were not neurovirulent in 3- to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.

Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE).

A yellow fever virus (YFV)/Japanese encephalitis virus (JEV) chimera in which the structural proteins prM and E of YFV 17D are replaced with those of the JEV SA14-14-2 vaccine strain is under evaluation as a candidate vaccine against Japanese encephalitis. The chimera (YFV/JEV SA14-14-2, or ChimeriVax-JE) is less neurovirulent than is YFV 17D vaccine in mouse and nonhuman primate models (F. Guirakhoo et al., Virology 257:363-372, 1999; T. P. Monath et al., Vaccine 17:1869-1882, 1999). Attenuation depends on the presence of the JEV SA14-14-2 E protein, as shown by the high neurovirulence of an analogous YFV/JEV Nakayama chimera derived from the wild JEV Nakayama strain (T. J. Chambers, A. Nestorowicz, P. W. Mason, and C. M. Rice, J. Virol. 73:3095-3101, 1999). Ten amino acid differences exist between the E proteins of ChimeriVax-JE and the YFV/JEV Nakayama virus, four of which are predicted to be neurovirulence determinants based on various sequence comparisons. To identify residues that are involved in attenuation, a series of intratypic YFV/JEV chimeras containing either single or multiple amino acid substitutions were engineered and tested for mouse neurovirulence. Reversions in at least three distinct clusters were required to restore the neurovirulence typical of the YFV/JEV Nakayama virus. Different combinations of cluster-specific reversions could confer neurovirulence; however, residue 138 of the E protein (E(138)) exhibited a dominant effect. No single amino acid reversion produced a phenotype significantly different from that of the ChimeriVax-JE parent. Together with the known genetic stability of the virus during prolonged cell culture and mouse brain passage, these findings support the candidacy of this experimental vaccine as a novel live-attenuated viral vaccine against Japanese encephalitis.

Phenotypic and molecular analyses of yellow fever 17DD vaccine viruses associated with serious adverse events in Brazil.

The yellow fever (YF) 17D virus is one of the most successful vaccines developed to data. Its use has been estimated to be over 400 million doses with an excellent record of safety. In the past 3 years, yellow fever vaccination was intensified in Brazil in response to higher risk of urban outbreaks of the disease. Two fatal adverse events temporally associated with YF vaccination were reported. Both cases had features similar to yellow fever disease, including hepatitis and multiorgan failure. Two different lots of YF 17DD virus vaccine were administered to the affected patients and also to hundreds of thousands of other individuals without any other reported serious adverse events. The lots were prepared from the secondary seed, which has been in continuous use since 1984. Nucleotide sequencing revealed minor variations at some nucleotide positions between the secondary seed lot virus and the virus isolates from patients; these differences were not consistent across the isolates, represented differences in the relative amount of each nucleotide in a heterogeneous position, and did not result in amino acid substitutions. Inoculation of rhesus monkeys with the viruses isolated from the two patients by the intracerebral (ic) or intrahepatic (ih) route caused minimal viremia and no clinical signs of infection or alterations in laboratory markers. Central nervous system histological scores of rhesus monkeys inoculated ic were within the expected range, and there were no histopathological lesions in animals inoculated ih. Altogether, these results demonstrated the genetic stability and attenuated phenotype of the viruses that caused fatal illness in the two patients. Therefore, the fatal adverse events experienced by the vaccinees are related to individual, genetically determined host factors that regulate cellular susceptibility to yellow fever virus. Such increased susceptibility, resulting in clinically overt disease expression, appears to be extremely rare.

West Nile virus vaccine.

Within the past 5 years, West Nile encephalitis has emerged as an important disease of humans and horses in Europe. In 1999, the disease appeared for the first time in the northeastern United States. West Nile virus (a mosquito-borne flavivirus) has flourished in the North American ecosystem and is expected to expand its geographic range. In this review, the rationale for a human and veterinary vaccine is presented and a novel approach for rapid development of a molecularly-defined, live, attenuated vaccine is described. The technology (ChimeriVax) is applicable to the development of vaccines against all flaviviruses, and products against Japanese encephalitis (a close relative of West Nile) and dengue are in or are nearing clinical trials, respectively. ChimeriVax vaccines utilize the safe and effective vaccine against the prototype flavivirus -yellow fever 17D- as a live vector. Infectious clone technology is used to replace the genes encoding the pre-membrane (prM) and envelope (E) protein of yellow fever 17D vaccine with the corresponding genes of the target virus (e.g., West Nile). The resulting chimeric virus contains the antigens responsible for protection against West Nile but retains the replication efficiency of yellow fever 17D. The ChimeriVax technology is well-suited to the rapid development of a West Nile vaccine, and clinical trials could begin as early as mid-2002. Other approaches to vaccine development are briefly reviewed. The aim of this brief review is to describe the features of West Nile encephalitis, a newly introduced infectious disease affecting humans, horses and wildlife in the United States; the rationale for rapid development of vaccines; and approaches to the development of vaccines against the disease.

Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates.

A chimeric yellow fever (YF)-dengue type 2 (dengue-2) virus (ChimeriVax-D2) was constructed using a recombinant cDNA infectious clone of a YF vaccine strain (YF 17D) as a backbone into which we inserted the premembrane (prM) and envelope (E) genes of dengue-2 virus (strain PUO-218 from a case of dengue fever in Bangkok, Thailand). The chimeric virus was recovered from the supernatant of Vero cells transfected with RNA transcripts and amplified once in these cells to yield a titer of 6.3 log(10) PFU/ml. The ChimeriVax-D2 was not neurovirulent for 4-week-old outbred mice inoculated intracerebrally. This virus was evaluated in rhesus monkeys for its safety (induction of viremia) and protective efficacy (induction of anti-dengue-2 neutralizing antibodies and protection against challenge). In one experiment, groups of non-YF-immune monkeys received graded doses of ChimeriVax-D2; a control group received only the vaccine diluents. All monkeys (except the control group) developed a brief viremia and showed no signs of illness. Sixty-two days postimmunization, animals were challenged with 5.0 log(10) focus forming units (FFU) of a wild-type dengue-2 virus. No viremia (<1.7 log(10) FFU/ml) was detected in any vaccinated group, whereas all animals in the placebo control group developed viremia. All vaccinated monkeys developed neutralizing antibodies in a dose-dependent response. In another experiment, viremia and production of neutralizing antibodies were determined in YF-immune monkeys that received either ChimeriVax-D2 or a wild-type dengue-2 virus. Low viremia was detected in ChimeriVax-D2-inoculated monkeys, whereas all dengue-2-immunized animals became viremic. All of these animals were protected against challenge with a wild-type dengue-2 virus, whereas all YF-immune monkeys and nonimmune controls became viremic upon challenge. Genetic stability of ChimeriVax-D2 was assessed by continuous in vitro passage in VeroPM cells. The titer of ChimeriVax-D2, the attenuated phenotype for 4-week-old mice, and the sequence of the inserted prME genes were unchanged after 18 passages in Vero cells. The high replication efficiency, attenuation phenotype in mice and monkeys, immunogenicity and protective efficacy, and genomic stability of ChimeriVax-D2 justify it as a novel vaccine candidate to be evaluated in humans.

Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys.

ChimeriVax-JE is a live, attenuated recombinant virus prepared by replacing the genes encoding two structural proteins (prM and E) of yellow fever 17D virus with the corresponding genes of an attenuated strain of Japanese encephalitis virus (JE), SA14-14-2 (T. J. Chambers et al., J. Virol. 73:3095-3101, 1999). Since the prM and E proteins contain antigens conferring protective humoral and cellular immunity, the immune response to vaccination is directed principally at JE. The prM-E genome sequence of the ChimeriVax-JE in diploid fetal rhesus lung cells (FRhL, a substrate acceptable for human vaccines) was identical to that of JE SA14-14-2 vaccine and differed from sequences of virulent wild-type strains (SA14 and Nakayama) at six amino acid residues in the envelope gene (E107, E138, E176, E279, E315, and E439). ChimeriVax-JE was fully attenuated for weaned mice inoculated by the intracerebral (i.c.) route, whereas commercial yellow fever 17D vaccine (YF-Vax) caused lethal encephalitis with a 50% lethal dose of 1.67 log(10) PFU. Groups of four rhesus monkeys were inoculated by the subcutaneous route with 2.0, 3.0, 4.0, and 5. 0 log(10) PFU of ChimeriVax-JE. All 16 monkeys developed low viremias (mean peak viremia, 1.7 to 2.1 log(10) PFU/ml; mean duration, 1.8 to 2.3 days). Neutralizing antibodies appeared between days 6 and 10; by day 30, neutralizing antibody responses were similar across dose groups. Neutralizing antibody titers to the homologous (vaccine) strain were higher than to the heterologous wild-type JE strains. All immunized monkeys and sham-immunized controls were challenged i.c. on day 54 with 5.2 log(10) PFU of wild-type JE. None of the immunized monkeys developed viremia or illness and had mild residual brain lesions, whereas controls developed viremia, clinical encephalitis, and severe histopathologic lesions. Immunized monkeys developed significant (>/=4-fold) increases in serum and cerebrospinal fluid neutralizing antibodies after i.c. challenge. In a standardized test for neurovirulence, ChimeriVax-JE and YF-Vax were compared in groups of 10 monkeys inoculated i.c. and analyzed histopathologically on day 30. Lesion scores in brains and spinal cord were significantly higher for monkeys inoculated with YF-Vax. ChimeriVax-JE meets preclinical safety and efficacy requirements for a human vaccine; it appears safer than yellow fever 17D vaccine but has a similar profile of immunogenicity and protective efficacy.

Growth characteristics of the chimeric Japanese encephalitis virus vaccine candidate, ChimeriVax-JE (YF/JE SA14--14--2), in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes.

The Japanese encephalitis (JE) virus vaccine candidate, ChimeriVax-JE, which consists of a yellow fever (YF) 17D virus backbone containing the prM and E genes from the JE vaccine strain JE SA14--14--2, exhibits restricted replication in non-human primates, producing only a low-level viremia following peripheral inoculation. Although this reduces the likelihood that hematophagous insects could become infected by feeding on a vaccinated host, it is prudent to investigate the replication kinetics of the vaccine virus in mosquito species that are known to vector the viruses from which the chimera is derived. In this study ChimeriVax-JE virus was compared to its parent viruses, as well as to wild-type JE virus, for its ability to replicate in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes. Individual mosquitoes were exposed to the viruses by oral ingestion of a virus-laden blood meal or by intrathoracic (IT) virus inoculation. ChimeriVax-JE virus did not replicate following ingestion by any of the three mosquito species. Additionally, replication was not detected after IT inoculation of ChimeriVax-JE in the primary JE virus vector, Cx. tritaeniorhynchus. ChimeriVax-JE exhibited moderate growth following IT inoculation into Ae. aegypti and Ae. albopictus, reaching titers of 3.6-5.0 log(10) PFU/mosquito. There was no change in the virus genotype associated with replication in mosquitoes. Similar results were observed in mosquitoes of all three species that were IT inoculated or had orally ingested the YF 17D vaccine virus. In contrast, all mosquitoes either IT inoculated with or orally fed wild-type and vaccine JE viruses became infected, reaching maximum titers of 5.4-7.3 log(10) PFU/mosquito. These results indicate that ChimeriVax-JE virus is restricted in its ability to infect and replicate in these mosquito vectors. The low viremia caused by ChimeriVax-JE in primates and poor infectivity for mosquitoes are safeguards against secondary spread of the vaccine virus.

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus.

HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka approximately 1x10(9) M-1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection.

Immunogenicity, genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-Japanese encephalitis virus (ChimeriVax-JE) as a live, attenuated vaccine candidate against Japanese encephalitis.

Yellow fever (YF) 17D vaccine virus, having a 60-year history of safe and effective use, is an ideal vector to deliver heterologous genes from other medically important flaviviruses. A chimeric YF/Japanese encephalitis (JE) virus (ChimeriVax-JE virus) was constructed by insertion of the premembrane and envelope (prME) genes of an attenuated human vaccine strain (SA14-14-2) of Japanese encephalitis (JE) virus between core and nonstructural (NS) genes of a YF 17D infectious clone. The virus grew to high titers in cell cultures and was not neurovirulent for 3- to 4-week-old mice at doses /=10(3) pfu of ChimeriVax-JE virus were solidly protected against intraperitoneal challenge with a virulent JE virus. Genetic stability of the chimera was assessed by sequential passages in cell cultures or in mouse brain. All attenuating residues and the avirulent phenotype were preserved after 18 passages in cell cultures or 6 passages in mouse brains.

Recombinant, chimaeric live, attenuated vaccine (ChimeriVax) incorporating the envelope genes of Japanese encephalitis (SA14-14-2) virus and the capsid and nonstructural genes of yellow fever (17D) virus is safe, immunogenic and protective in non-human primates.

Yellow fever 17D virus, a safe and effective live, attenuated vaccine, was used as a vector for genes encoding the protective antigenic determinants of a heterologous member of the genus Flavivirus, Japanese encephalitis (JE) virus, the leading cause of acute viral central nervous system infection and death throughout Asia. The viral envelope (prM and E) genes of a full-length cDNA clone of YF 17D virus were replaced with the corresponding genes of JE SA14-14-2, a strain licensed as a live, attenuated vaccine in China. Full-length RNA transcripts of the YF/JE chimaera were used to transfect Vero cells. The progeny virus (named 'ChimeriVax-JE'), was used to define safety after intracerebral (i.c.) inoculation of rhesus monkeys. Monkeys (N = 3) inoculated with a high dose (6.6 log10 pfu) developed a brief viremia, showed no signs of illness, developed high titers of anti-JE neutralizing antibody, and had minimal brain and spinal cord lesion scores according to criteria specified in the WHO monkey neurovirulence test. A control group of 3 monkeys that received a lower dose (4.2 log10 pfu) of commercial YF 17D vaccine had slightly higher lesion scores. To develop a lethal monkey model of JE for vaccine protection tests, we inoculated groups of monkeys i.c. or intranasally (i.n.) with a JE virus strain found to be highly neurovirulent and neuroinvasive for mice. Monkeys inoculated i.c., but not i.n., developed severe encephalitis after an incubation period of 8-13 days. The ChimeriVax-JE virus was passed in a cell line acceptable for human use (diploid fetal rhesus lung) and 4.3 or 5.3 log10 pfu were inoculated into groups of 3 monkeys by the subcutaneous route. All 6 animals developed brief viremias (peak titer < 2.0 log10 pfu/ml) and subsequently had anti-JE but no yellow fever neutralizing antibodies. On day 64, the monkeys were challenged i.c. with 5.5 log10 pfu of virulent JE virus. The immunized animals had no detectable viremia post-challenge, whereas 4 unimmunized controls became viremic. Only 1 of 6 (17%) vaccinated monkeys but 4 of 4 (100%) unvaccinated controls developed encephalitis. Histopathological examination 30 days after challenge confirmed that the protected, immunized animals had no or minimal evidence of encephalitis. These data demonstrated the ability of the ChimeriVax-JE to induce a rapid humoral immune response and to protect against a very severe, direct intracerebral virus challenge. Target areas of neuronal damage and inflammation in monkeys infected IC with wild-type JE, the chimaeric virus and YF 17D were similar, indicating that the histopathological scoring system used for the WHO yellow fever monkey neurovirulence test will be applicable to control testing of chimaeric seed viruses and vaccines.

Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection.

Intranasal (i.n.) delivery of antigen can be highly effective for generating circulating and secretory antibody responses. Mice were immunized i.n. with two antigens, human IgA, and Helicobacter pylori urease in the presence or absence of mucosal adjuvant. To restrict antigen delivery to the upper airways, protein solutions were administered in a small volume without anesthesia. Repeated daily i.n. administration of antigen without adjuvant elicited high levels of specific IgG in serum and IgA in serum, saliva, and feces. Once weekly i.n. immunization with co-administration of cholera toxin or Escherichia coli heat-labile toxin as adjuvant elicited somewhat lower levels of antibody to urease. When challenged with Helicobacter felis, only mice immunized with urease in the presence of adjuvant were protected against gastric infection.

Cloning, expression and functional activities of a single chain antibody fragment directed to fusion protein of respiratory syncytial virus.

BACKGROUND: HNK20 is a murine IgA which is currently being investigated in clinical trials against respiratory syncytial virus (RSV) infections in infants and young children. OBJECTIVE: To produce a single chain antibody fragment (scFv) from HNK20 hybridoma cells and assess its functional activities in vitro and in vivo (mouse model). STUDY DESIGN: The V regions of heavy and light chains were cloned and linked by a sequence encoding for (Gly4 Ser)3 and expressed in Escherichia coli. RESULTS: Over 100 mg/l of the HNK20-scFv was produced in shake flasks after induction with isopropyl (beta-D-thiogalactopyranoside (IPTG). ScFv was purified under native conditions on a Ni2+ affinity column and migrated as a single band of 34 kDa on sodium dodecyl sulfate (SDS)-gels. ScFv demonstrated similar affinity as its parent IgA molecule, neutralized RSV in vitro and significantly reduced RSV titers in lungs of mice when administered intranasally shortly before or a day after RSV challenge. CONCLUSION: It is possible that this scFv or its derivatives, when applied by intranasal or pulmonary route, will be useful for treatment of RSV infections in infants and young children.

Adaptation of bluetongue virus in mosquito cells results in overexpression of NS3 proteins and release of virus particles.

Adaptation of bluetongue virus (BTV) to grow in mosquito cells (C6/36) resulted in overexpression of two non-structural proteins (NS3 and NS3a) in infected cells. These proteins also co-purified with BTV particles and were dissociated from the virions upon treatment with an anionic detergent. The expression was not host dependent, since back inoculation of the adapted virus into mammalian cell cultures also resulted in a significant overexpression of these proteins. The BTV-C6/36 produced smaller plaques in Vero cells compared with the parent strain. This is the first report which demonstrates a high level of NS3/NS3a expression in infected cells and subsequent release of infectious BTV particles into the supernatant.

The envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites.

We have previously isolated and characterized two dengue (DEN) 2 viruses mutant in their fusion-from-within (FFWI) phenotype in the insect cell line C6/36. Both viruses lost a potential glycosylation site (Asn-153) in the envelope (E) glycoprotein. To determine whether the change in FFWI phenotype was due to a change in E-glycoprotein glycosylation, we characterized the patterns of glycosylation on the E-glycoprotein of wild-type DEN 1 and DEN 2 viruses. The E-glycoproteins were isolated from purified virus grown in Aedes albopictus C6/36 cells, by use of high-performance size-exclusion chromatography. The tryptic maps of wild-type glycosylated and enzymatically (PNGase F) deglycosylated E-glycoproteins were compared by reverse-phase high-performance liquid chromatography. The DEN 1 virus E-glycoprotein was found to have two peaks in the tryptic map that exhibited shifts after deglycosylation, whereas the DEN 2 virus E-glycoprotein had only one. Besides the potential glycosylation site at Asn-153, both DEN 1 and DEN 2 virus E-glycoproteins have another potential site located at Asn-67. Amino-terminal sequencing of the shifted peaks revealed that DEN 2 virus E-glycoprotein is glycosylated only at Asn-67; however, DEN 1 virus E-glycoprotein is glycosylated at both Asn-67 and Asn-153. These DEN virus serotypes are thus heterogeneous in their use of glycosylation sites. We also determined by a lectin-binding assay that the attached carbohydrates for both viruses were likely to be of the high-mannose type.

The interactions of the flavivirus envelope proteins: implications for virus entry and release.

Viral membrane proteins play an important role in the assembly and disassembly of enveloped viruses. Oligomerization and proteolytic cleavage events are involved in controlling the functions of these proteins during virus entry and release. Using tick-borne encephalitis virus as a model we have studied the role of the flavivirus envelope proteins E and prM/M in these processes. Experiments with acidotropic agents provide evidence that the virus is taken up by receptor-mediated endocytosis and that the acidic pH in endosomes plays an important role for virus entry. The envelope glycoprotein E undergoes irreversible conformational changes at acidic pH, as indicated by the loss of several monoclonal antibody-defined epitopes, which coincide with the viral fusion activity in vitro. Sedimentation analysis reveals that these conformational changes lead to aggregation of virus particles, apparently by the exposure of hydrophobic sequence elements. None of these features are exhibited by immature virions containing E and prM rather than E and M. Detergent solubilization, sedimentation, and crosslinking experiments provide evidence that prM forms a complex with protein E which prevents the conformational changes necessary for fusion activity. The functional role of prM before its endoproteolytic cleavage by a cellular protease thus seems to be the protection of protein E from acid-inactivation during its passage through acidic trans Golgi vesicles in the course of virus release.

Selection and partial characterization of dengue 2 virus mutants that induce fusion at elevated pH.

Two types of dengue (DEN) 2 virus mutants were selected either by repeated exposure to acidic pH (acid mutant, AM), or by the addition of ammonium chloride to Aedes albopictus C6/36 cells prior to and during viral infection (fusion mutant, FM). Both mutants grew more slowly than the parent strain and induced smaller plaques in Vero cells. The 50% fusion from within index for both mutants occurred at least 0.65 pH units higher than with the wild-type DEN virus. A single amino acid substitution (Asn-153 to Asp) was found in the envelope (E)-glycoprotein of the AM virus. Three amino acid substitutions were detected on the E-glycoprotein of the FM virus: Ile-6 to Met, Asn-134 to Ser, and Asn-153 to Tyr. No mutations were found in the precursor to the membrane protein, prM. The DEN virus E-glycoprotein has two potential glycosylation sites: Asn-67 and Asn-153. The loss of the potential glycosylation site at Asn-153 or the change in the chemical characteristics resultant from the amino acid substitutions in both mutants implicates these regions of the E-glycoprotein in virus-mediated membrane fusion.

The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein.

To study the role of the precursor to the membrane protein (prM) in flavivirus maturation, we inhibited the proteolytic processing of the Murray Valley encephalitis (MVE) virus prM to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. Viruses purified from supernatants of ammonium chloride-treated cells contained prM protein and were unable to fuse C6/36 mosquito cells from without. When ammonium chloride was removed from the cells, both the processing of prM and the fusion activity of the purified viruses were partially restored. By using monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of MVE virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prM-containing MVE virus particles. Amino-terminal sequencing of proteolytic fragments of the E protein which were reactive with sequence-specific peptide antisera or MAb enabled us to estimate the site of the E protein interacting with the prM to be within amino acids 200 to 327. Since prM-containing viruses were up to 400-fold more resistant to a low pH environment, we conclude that the E-prM interaction might be necessary to protect the E protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway.