Search

ChimeriVax-WN02 is a novel live-attenuated West Nile virus (WNV) vaccine containing modified WNV premembrane (prM) and envelope (E) sequences inserted into the yellow fever 17D vaccine genome. We investigated the induction and evolution of CD8(+) T cell responses to a WNV envelope epitope, which is a dominant target in naturally infected HLA-A*02-positive individuals. WNV epitope-specific CD8(+) T cells were detected by HLA tetramer staining in 22 of 23 donors tested, with peak frequencies occurring between days 14 and 28. WNV epitope-specific T cells evolved from an effector phenotype to a long-lived memory phenotype. In the majority of donors, CD8(+) T cells were able to lyse targets expressing WNV envelope protein and produced macrophage inflammatory protein 1ß, interferon γ, and/or tumor necrosis factor α following envelope peptide stimulation. WNV E-specific CD8(+) T cell responses were detected for up to 1 year after vaccination. The evolution of this WNV-specific T cell response is similar to that observed in established, highly immunogenic vaccines. [ABSTRACT FROM AUTHOR]

Flavivirus vaccines based on ChimeriVax technology contain the nonstructural genes of the yellow fever vaccine and the premembrane and envelope genes of heterologous flaviviruses, such as Japanese encephalitis and West Nile viruses. These chimeric vaccines induce both humoral and cell-mediated immunity. Mice were vaccinated with yellow fever, chimeric Japanese encephalitis virus (YF/JE), or chimeric West Nile virus (YF/WN) vaccines, followed by a secondary homologous or heterologous vaccination; the hierarchy and function of CD8(+) T cell responses to a variable envelope epitope were then analyzed and compared with those directed against a conserved immunodominant yellow fever virus NS3 epitope. Sequential vaccination with heterologous chimeric flaviviruses generated a broadly cross-reactive CD8(+) T cell response dependent on both the sequence of infecting viruses and epitope variant. The enhanced responses to variant epitopes after heterologous vaccination were not related to preexisting antibody or to higher virus titers. These results demonstrate that the sequence of vaccination affects the expansion of cross-reactive CD8(+) T cells after heterologous chimeric flavivirus challenge. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 ow 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 ali 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. [ABSTRACT FROM AUTHOR]

Antibodies provide critical protective immunity against COVID-19, and the Fc-mediated effector functions and mucosal antibodies also contribute to the protection. To expand the characterization of humoral immunity stimulated by subunit protein-peptide COVID-19 vaccine UB-612, preclinical studies in non-human primates were undertaken to investigate mucosal secretion and the effector functionality of vaccine-induced antibodies in antibody-dependent monocyte phagocytosis (ADMP) and antibody-dependent NK cell activation (ADNKA) assays. In cynomolgus macaques, UB-612 induced potent serum-neutralizing, RBD-specific IgG binding, ACE2 binding-inhibition antibodies, and antibodies with Fc-mediated effector functions in ADMP and ADNKA assays. Additionally, immunized animals developed mucosal antibodies in bronchoalveolar lavage fluids (BAL). The level of mucosal or serum ADMP and ADNKA antibodies was found to be UB-612 dose-dependent. Our results highlight that the novel subunit UB-612 vaccine is a potent B-cell immunogen inducing polyfunctional antibody responses contributing to anti-viral immunity and vaccine efficacy.

The development of safe and effective vaccines to respond to COVID-19 pandemic/endemic remains a priority. We developed a novel subunit protein-peptide COVID-19 vaccine candidate (UB-612) composed of: (i) receptor binding domain of SARS-CoV-2 spike protein fused to a modified single-chain human IgG1 Fc; (ii) five synthetic peptides incorporating conserved helper and cytotoxic T lymphocyte (Th/CTL) epitopes derived from SARS-CoV-2 structural proteins (three from S2 subunit, one from membrane and one from nucleocapsid), and one universal Th peptide; (iii) aluminum phosphate as adjuvant. The immunogenicity and protective immunity induced by UB-612 vaccine were evaluated in four animal models: Sprague-Dawley rats, AAV-hACE2 transduced BALB/c mice, rhesus and cynomolgus macaques. UB-612 vaccine induced high levels of neutralizing antibody and T-cell responses, in all animals. The immune sera from vaccinated animals neutralized the SARS-CoV-2 original wild-type strains and multiple variants of concern, including Delta and Omicron. The vaccination significantly reduced viral loads, lung pathology scores, and disease progression after intranasal and intratracheal challenge with SARS-CoV-2 in mice, rhesus and cynomolgus macaques. UB-612 has been tested in primary regimens in Phase 1 and Phase 2 clinical studies and is currently being evaluated in a global pivotal Phase 3 clinical study as a single dose heterologous booster.

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has caused high rates of breakthrough infections in those previously vaccinated with ancestral strain coronavirus disease 2019 (COVID-19) vaccines. Here, we demonstrate that a booster dose of UB-612 vaccine candidate delivered 7-9 months after primary vaccination increased neutralizing antibody levels by 131-, 61-, and 49-fold against ancestral SARS-CoV-2 and the Omicron BA.1 and BA.2 variants, respectively. Based on the receptor-binding domain protein binding antibody responses, the UB-612 third-dose booster may lead to an estimated approximately 95% efficacy against symptomatic COVID-19 caused by the ancestral strain. Our results support UB-612 as a potential potent booster against current and emerging SARS-CoV-2 variants., Competing Interests: Potential conflicts of interest. F. G., S. W., L. W., M. M. H., T. M., and A. R. are employees of Vaxxinity Inc (Dallas, Texas). C. Y. W., W.-J. P., H.-K. K., and H. L. are employees of United Biomedical Inc Asia (Hsinchu, Taiwan). All other authors report no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A new vectored vaccine MVA-VLP-SUDV was generated against Sudan ebolavirus (SUDV) combining the advantages of the immunogenicity of a live attenuated vaccine vector (Modified Vaccinia Ankara, MVA) with the authentic conformation of virus-like particles (VLPs). The vaccine expresses minimal components to generate self-assembling VLPs in the vaccinee: the envelope glycoprotein GP and the matrix protein VP40. Guinea pigs vaccinated with one dose of MVA-VLP-SUDV generated SUDV-specific binding and neutralizing antibody responses as well as Fc-mediated protective effects. These responses were boosted by a second vaccine dose. All vaccinated animals which received either one or two vaccine doses were protected from death and disease symptoms following challenge with a lethal dose of SUDV. These data demonstrate single dose protection and potency of the MVA-VLP platform for use in emergency situations to contain outbreaks., (© 2022. The Author(s).)

BackgroundThe Delta and Omicron variants of SARS-CoV-2 are currently responsible for breakthrough infections due to waning immunity. We report phase I/II trial results of UB-612, a multitope subunit vaccine containing S1-RBD-sFc protein and rationally designed promiscuous peptides representing sarbecovirus conserved helper T cell and cytotoxic T lymphocyte epitopes on the nucleocapsid (N), membrane (M), and spike (S2) proteins.MethodWe conducted a phase I primary 2-dose (28 days apart) trial of 10, 30, or 100 μg UB-612 in 60 healthy young adults 20 to 55 years old, and 50 of them were boosted with 100 μg of UB-612 approximately 7 to 9 months after the second dose. A separate placebo-controlled and randomized phase II study was conducted with 2 doses of 100 μg of UB-612 (n = 3,875, 18-85 years old). We evaluated interim safety and immunogenicity of phase I until 14 days after the third (booster) dose and of phase II until 28 days after the second dose.ResultsNo vaccine-related serious adverse events were recorded. The most common solicited adverse events were injection site pain and fatigue, mostly mild and transient. In both trials, UB-612 elicited respective neutralizing antibody titers similar to a panel of human convalescent sera. The most striking findings were long-lasting virus-neutralizing antibodies and broad T cell immunity against SARS-CoV-2 variants of concern (VoCs), including Delta and Omicron, and a strong booster-recalled memory immunity with high cross-reactive neutralizing titers against the Delta and Omicron VoCs.ConclusionUB-612 has presented a favorable safety profile, potent booster effect against VoCs, and long-lasting B and broad T cell immunity that warrants further development for both primary immunization and heterologous boosting of other COVID-19 vaccines.Trial RegistrationClinicalTrials.gov: NCT04545749, NCT04773067, and NCT04967742.FundingUBI Asia, Vaxxinity Inc., and Taiwan Centers for Disease Control, Ministry of Health and Welfare.

We introduce a new vaccine platform against Marburg virus (MARV) combining the advantages of the immunogenicity of a highly attenuated vaccine vector (Modified Vaccinia Ankara, MVA) with the authentic conformation of virus-like particles (VLPs). Our vaccine, MVA-MARV-VLP, expresses the minimal components of MARV VLPs: the envelope glycoprotein GP and the matrix protein VP40. Electron microscopy confirmed self-assembly and budding of VLPs from infected cells. Prime/boost vaccination of guinea pigs with MVA-MARV-VLP-elicited MARV-specific binding and neutralizing antibody responses. Vaccination also induced Fc-mediated innate immune effector functions including activation of NK cells and antibody-dependent phagocytosis by neutrophils and monocytes. Inoculation of vaccinated animals with guinea pig-adapted MARV demonstrated 100% protection against death and disease with no viremia. Therefore, our vaccine platform, expressing two antigens resulting in assembly of VLPs in the native conformation in vaccinated hosts, can be used as a potent vaccine against MARV., Competing Interests: COMPETING INTERESTSA.D., M.J.C., and F.G. has competing interest as being a full time employee of GeoVax. D.C.M., M.M., B.M.G., G.A., and A.B. declare that there are no competing interests., (© The Author(s) 2020.)

Lassa fever surpasses Ebola, Marburg, and all other hemorrhagic fevers except Dengue in its public health impact. Caused by Lassa virus (LASV), the disease is a scourge on populations in endemic areas of West Africa, where reported incidence is higher. Here, we report construction, characterization, and preclinical efficacy of a novel recombinant vaccine candidate GEO-LM01. Constructed in the Modified Vaccinia Ankara (MVA) vector, GEO-LM01 expresses the glycoprotein precursor (GPC) and zinc-binding matrix protein (Z) from the prototype Josiah strain lineage IV. When expressed together, GP and Z form Virus-Like Particles (VLPs) in cell culture. Immunogenicity and efficacy of GEO-LM01 was tested in a mouse challenge model. A single intramuscular dose of GEO-LM01 protected 100% of CBA/J mice challenged with a lethal dose of ML29, a Mopeia/Lassa reassortant virus, delivered directly into the brain. In contrast, all control animals died within one week. The vaccine induced low levels of antibodies but Lassa-specific CD4 + and CD8 + T cell responses. This is the first report showing that a single dose of a replication-deficient MVA vector can confer full protection against a lethal challenge with ML29 virus.

Ebola virus (EBOV), isolate Makona, was the causative agent of the West African epidemic devastating predominantly Guinea, Liberia and Sierra Leone from 2013-2016. While several experimental vaccine and treatment approaches have been accelerated through human clinical trials, there is still no approved countermeasure available against this disease. Here, we report the construction and preclinical efficacy testing of a novel recombinant modified vaccinia Ankara (MVA)-based vaccine expressing the EBOV-Makona glycoprotein GP and matrix protein VP40 (MVA-EBOV). GP and VP40 form EBOV-like particles and elicit protective immune responses. In this study, we report 100% protection against lethal EBOV infection in guinea pigs after prime/boost vaccination with MVA-EBOV. Furthermore, this MVA-EBOV protected macaques from lethal disease after a single dose or prime/boost vaccination. The vaccine elicited a variety of antibody responses to both antigens, including neutralizing antibodies and antibodies with antibody-dependent cellular cytotoxic activity specific for GP. This is the first report that a replication-deficient MVA vector can confer full protection against lethal EBOV challenge after a single dose vaccination in macaques.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has rapidly extended its geographic range around the world. Its association with abnormal fetal brain development, sexual transmission, and lack of a preventive vaccine have constituted a global health concern. Designing a safe and effective vaccine requires significant caution due to overlapping geographical distribution of ZIKV with dengue virus (DENV) and other flaviviruses, possibly resulting in more severe disease manifestations in flavivirus immune vaccinees such as Antibody-Dependent Enhancement (ADE, a phenomenon involved in pathogenesis of DENV, and a risk associated with ZIKV vaccines using the envelope proteins as immunogens). Here, we describe the development of an alternative vaccine strategy encompassing the expression of ZIKV non-structural-1 (NS1) protein from a clinically proven safe, Modified Vaccinia Ankara (MVA) vector, thus averting the potential risk of ADE associated with structural protein-based ZIKV vaccines. A single intramuscular immunization of immunocompetent mice with the MVA-ZIKV-NS1 vaccine candidate provided robust humoral and cellular responses, and afforded 100% protection against a lethal intracerebral dose of ZIKV (strain MR766). This is the first report of (i) a ZIKV vaccine based on the NS1 protein and (ii) single dose protection against ZIKV using an immunocompetent lethal mouse challenge model.

Although first isolated almost 70 years ago, Zika virus (ZIKV; Flavivirus, Flaviviridae ) has only recently been associated with significant outbreaks of disease in humans. Several severe ZIKV disease manifestations have also been recently documented, including fetal malformations, such as microcephaly, and Guillain-Barré syndrome in adults. Although principally transmitted by mosquitoes, sexual transmission of ZIKV has been documented. Recent publications of several interferon receptor knockout mouse models have demonstrated ZIKV-induced disease. Herein, outbred immunocompetent CD-1/ICR adult mice were assessed for susceptibility to disease by intracranial (i.c.) and intraperitoneal (i.p.) inoculation with the Ugandan prototype strain (MR766; African genotype), a low-passage Senegalese strain (DakAr41524; African genotype) and a recent ZIKV strain isolated from a traveler infected in Puerto Rico (PRVABC59; Asian genotype). Morbidity was not observed in mice inoculated by the i.p. route with either MR766 or PRVABC59 for doses up to 6 log 10 PFU. In contrast, CD-1/ICR mice inoculated i.c. with the MR766 ZIKV strain exhibited an 80-100% mortality rate that was age independent. The DakAr41524 strain delivered by the i.c route caused 30% mortality, and the Puerto Rican ZIKV strain failed to elicit mortality but did induce a serum neutralizing immune response in 60% of mice. These data provide a potential animal model for assessing neurovirulence determinants of different ZIKV strains as well as a potential immunocompetent challenge model for assessing protective efficacy of vaccine candidates.

Subunit vaccines for prevention of congenital cytomegalovirus (CMV) infection based on glycoprotein B (gB) and pp65 are in clinical trials, but it is unclear whether simultaneous vaccination with both antigens enhances protection. We undertook evaluation of a novel bivalent vaccine based on nonreplicating lymphocytic choriomeningitis virus (rLCMV) vectors expressing a cytoplasmic tail-deleted gB [gB(dCt)] and full-length pp65 from human CMV in mice. Immunization with the gB(dCt) vector alone elicited a comparable gB-binding antibody response and a superior neutralizing response to that elicited by adjuvanted subunit gB. Immunization with the pp65 vector alone elicited robust T cell responses. Comparable immunogenicity of the combined gB(dCt) and pp65 vectors with the individual monovalent formulations was demonstrated. To demonstrate proof of principle for a bivalent rLCMV-based HCMV vaccine, the congenital guinea pig cytomegalovirus (GPCMV) infection model was used to compare rLCMV vectors encoding homologs of pp65 (GP83) and gB(dCt), alone and in combination versus Freund's adjuvanted recombinant gB. Both vectors elicited significant immune responses, and no loss of gB immunogenicity was noted with the bivalent formulation. Combined vaccination with rLCMV-vectored GPCMV gB(dCt) and pp65 (GP83) conferred better protection against maternal viremia than subunit or either monovalent rLCMV vaccine. The bivalent vaccine also was significantly more effective in reducing pup mortality than the monovalent vaccines. In summary, bivalent vaccines with rLCMV vectors expressing gB and pp65 elicited potent humoral and cellular responses and conferred protection in the GPCMV model. Further clinical trials of LCMV-vectored HCMV vaccines are warranted., (Copyright © 2017 American Society for Microbiology.)

Background: Congenital cytomegalovirus infection can be life-threatening and often results in significant developmental deficits and/or hearing loss. Thus, there is a critical need for an effective anti-CMV vaccine., Objective: To determine the efficacy of replication-defective lymphocytic choriomeningitis virus (rLCMV) vectors expressing the guinea pig CMV (GPCMV) antigens, gB and pp65, in the guinea pig model of congenital CMV infection., Methods: Female Hartley strain guinea pigs were divided into three groups: Buffer control group (n = 9), rLCMV-gB group (n = 11), and rLCMV-pp65 (n = 11). The vaccines were administered three times IM at 1.54 × 10(6)FFU per dose at 21-day intervals. At two weeks after vaccination, the female guinea pigs underwent breeding. Pregnant guinea pigs were challenged SQ at ∼ 45-55 days of gestation with 1 × 10(5)PFU of GPCMV. Viremia in the dams, pup survival, weights of pups at delivery, and viral load in both dam and pup tissues were determined., Results: Pup survival was significantly increased in the LCMV-gB vaccine group. There was 23% pup mortality in the gB vaccine group (p = 0.044) and 26% pup mortality in the pp65 vaccine group (p = 0.054) compared to 49% control pup mortality. The gB vaccine induced high levels of gB binding and detectable neutralizing antibodies, reduced dam viremia, and significantly reduced viral load in dam tissues compared to control dams (p < 0.03). Reduced viral load and transmission in pups born to gB-vaccinated dams was observed compared to pups from pp65-vaccinated or control dams., Conclusions: The rLCMV-gB vaccine significantly improved pup survival and also increased pup weights and gestation time. The gB vaccine was also more effective at decreasing viral load in dams and pups and limiting congenital transmission. Thus, rLCMV vectors that express CMV antigens may be an effective vaccine strategy for congenital CMV infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.

Dengue viruses (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are major global health and growing medical problems. While a live-attenuated vaccine exists since decades against the prototype flavivirus, yellow fever virus (YFV), there is an urgent need for vaccines against dengue or West Nile diseases, and for improved vaccines against Japanese encephalitis. Live-attenuated chimeric viruses were constructed by replacing the genes coding for Premembrane (prM) and Envelope (E) proteins from YFV 17D vaccine strain with those of heterologous flaviviruses (ChimeriVax technology). This technology has been used to produce vaccine candidates for humans, for construction of a horse vaccine for West Nile fever, and as diagnostic reagents for dengue, Japanese encephalitis, West Nile and St. Louis encephalitis infections. This review focuses on human vaccines and their characterization from the early stages of research through to clinical development. Phenotypic and genetic properties and stability were examined, preclinical evaluation through in vitro or animal models, and clinical testing were carried out. Theoretical environmental concerns linked to the live and genetically modified nature of these vaccines have been carefully addressed. Results of the extensive characterizations are in accordance with the immunogenicity and excellent safety profile of the ChimeriVax-based vaccine candidates, and support their development towards large-scale efficacy trials and registration.

The plaque reduction neutralization test (PRNT) is a specific serological test used to identify and confirm arbovirus infection in diagnostic laboratories and monitor immunological protection in vaccine recipients. Wild-type (wt) viruses used in the PRNT may be difficult to grow and plaque titrate, such as the dengue viruses (DENV), and/or may require biosafety level 3 (BSL3) containment, such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). These requirements preclude their use in diagnostic laboratories with only BSL2 capacity. In addition, wt JEV falls under the jurisdiction of the select-agent program and can be used only in approved laboratories. The chimeric vaccine viruses ChimeriVax-WNV and -SLEV have previously been shown to elicit antibody reactivity comparable to that of parental wt WNV and SLEV. ChimeriVax viruses provide advantages for PRNT, as follows: they grow more rapidly than most wt flaviviruses, produce large plaques, require BSL2 conditions, and are not under select-agent restrictions. We evaluated the ChimeriVax-DENV serotype 1 (DENV1), -DENV2, -DENV3, -DENV4, and -JEV for use in PRNT on sera from DENV- and JEV-infected patients and from JEV vaccine recipients. Serostatus agreement was 100% between the ChimeriVax-DENV serotypes and wt prototype DENV and 97% overall with ChimeriVax-JEV compared to prototype Nakayama JEV, 92% in a subgroup of JEV vaccine recipients, and 100% in serum from encephalitis patients naturally infected with JEV. ChimeriVax-DENV and -JEV plaque phenotype and BSL2 requirements, combined with sensitive and specific reactivity, make them good substitutes for wt DENV and JEV in PRNT in public health diagnostic laboratories.

T-cell epitope variability is associated with viral immune escape and may influence the outcome of vaccination against the highly variable Japanese Encephalitis Virus (JEV). We computationally analyzed the ChimeriVax-JEV vaccine envelope sequence for T helper epitopes that are conserved in 12 circulating JEV strains and discovered 75% conservation among putative epitopes. Among non-identical epitopes, only minor amino acid changes that would not significantly affect HLA-binding were present. Therefore, in most cases, circulating strain epitopes could be restricted by the same HLA and are likely to stimulate a cross-reactive T-cell response. Based on this analysis, we predict no significant abrogation of ChimeriVax-JEV-conferred protection against circulating JEV strains.

Although the theoretical concern of genetic recombination has been raised related to the use of live attenuated flavivirus vaccines [Seligman, Gould, Lancet 2004;363:2073-5], it has little foundation [e.g., Monath TP, Kanesa-Thasan N, Guirakhoo F, Pugachev K, Almond J, Lang J, et al. Vaccine 2005;23:2956-8]. To investigate biological effects of recombination between a chimeric yellow fever (YF) 17D/Japanese encephalitis (JE) vaccine virus (ChimeriVax-JE) and a wild-type flavivirus Kunjin (KUN-cDNA), the prM-E envelope protein genes were swapped between the two viruses, resulting in new YF 17D/KUN(prM-E) and KUN/JE(prM-E) chimeras. The prM-E genes are easily exchangeable between flavivirues, and thus the exchange was expected to yield the most replication-competent chimeras, while other rationally designed recombinants would be more likely to be crippled or non-viable. The new chimeras proved highly attenuated in comparison with the KUN-cDNA parent, as judged by plaque size and growth kinetics in cell culture, low viremia in hamsters, and reduced neurovirulence/neuroinvasiveness in mice. These data provide strong experimental evidence that the potential of recombinants, should they ever emerge, to cause disease or spread (compete in nature with wild-type flaviviruses) would be indeed extremely low.

Numerous viruses of the Flaviviridae family, including dengue, yellow fever, Japanese encephalitis, and West Nile, cause significant disease in humans and animals. The structure and function of the molecular components of the flavivirus envelope are therefore of significant interest. To our knowledge, a membrane (M) protein mutation which affects the pH at which flavivirus particles are inactivated in vitro has never been reported. Here we show that substitution of proline for glutamine at residue M5 (MQ5P) of a Japanese encephalitis-yellow fever chimera (ChimeriVax-JE) increases its acid sensitivity in vitro by 0.3 pH units (i.e., increases the pH at which virus titer is reduced by 50% from 6.08 to 6.38). In addition, growth kinetics of this mutant virus are accelerated in Vero cells, while neurovirulence and neuroinvasiveness measured in a mouse model are unaffected. A possible interpretation of these observations is that M can modulate the envelope (E) protein function during cell infection.

Four chimeric yellow fever (YF) 17D-dengue (DEN) candidate vaccine viruses (ChimeriVax-DEN; Acambis, Cambridge, MA) were characterized in Aedes aegypti and Ae. albopictus mosquitoes collected from Thailand. The four vaccine viruses contained the relevant prM and E genes of wild-type dengue viruses (DENV; serotypes 1-4) substituted for the equivalent genes in the YF vaccine virus (17D) backbone. Each chimera conferred protection against the homologous DENV serotype; a tetravalent mix of all four chimeras stimulates an immune response against all serotypes. Field-collected mosquitoes from Thailand were fed on blood containing each of the viruses under study and held 21 days after infection. Infection and dissemination rates were based on antigen detection in the body or head tissues, respectively. All four wild-type DENV serotypes infected and disseminated, but the candidate vaccine viruses were highly attenuated in mosquitoes with respect to infection and especially with respect to dissemination. Considering the low level viremias anticipated in humans vaccinated with these viruses, it is predicted that the risks of infection and transmission by mosquitoes in nature is minimal.

Australian mosquitoes from which Japanese encephalitis virus (JEV) has been recovered (Culex annulirostris, Culex gelidus, and Aedes vigilax) were assessed for their ability to be infected with the ChimeriVax-JE vaccine, with yellow fever vaccine virus 17D (YF 17D) from which the backbone of ChimeriVax-JE vaccine is derived and with JEV-Nakayama. None of the mosquitoes became infected after being fed orally with 6.1 log(10) plaque-forming units (PFU)/mL of ChimeriVax-JE vaccine, which is greater than the peak viremia in vaccinees (mean peak viremia = 4.8 PFU/mL, range = 0-30 PFU/mL of 0.9 days mean duration, range = 0-11 days). Some members of all three species of mosquito became infected when fed on JEV-Nakayama, but only Ae. vigilax was infected when fed on YF 17D. The results suggest that none of these three species of mosquito are likely to set up secondary cycles of transmission of ChimeriVax-JE in Australia after feeding on a viremic vaccinee.

West Nile (WN) virus is an important cause of febrile exanthem and encephalitis. Since it invaded the U.S. in 1999, >19,000 human cases have been reported. The threat of continued epidemics has spurred efforts to develop vaccines. ChimeriVax-WN02 is a live, attenuated recombinant vaccine constructed from an infectious clone of yellow fever (YF) 17D virus in which the premembrane and envelope genes of 17D have been replaced by the corresponding genes of WN virus. Preclinical tests in monkeys defined sites of vaccine virus replication in vivo. ChimeriVax-WN02 and YF 17D had similar biodistribution but different multiplication kinetics. Prominent sites of replication were skin and lymphoid tissues, generally sparing vital organs. Viruses were cleared from blood by day 7 and from tissues around day 14. In a clinical study, healthy adults were inoculated with 5.0 log(10) plaque-forming units (PFU) (n = 30) or 3.0 log10 PFU (n = 15) of ChimeriVax-WN02, commercial YF vaccine (YF-VAX, n = 5), or placebo (n = 30). The incidence of adverse events in subjects receiving the vaccine was similar to that in the placebo group. Transient viremia was detected in 42 of 45 (93%) of ChimeriVax-WN02 subjects, and four of five (80%) of YF-VAX subjects. All subjects developed neutralizing antibodies to WN or YF, respectively, and the majority developed specific T cell responses. ChimeriVax-WN02 rapidly elicits strong immune responses after a single dose, and is a promising candidate warranting further evaluation for prevention of WN disease.

A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax-DEN2) in comparison to that of YF vaccine (YF-VAX). Forty-two healthy YF naïve adults randomly received a single dose of either ChimeriVax-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAX by the subcutaneous route (SC). To determine the effect of YF preimmunity on the ChimeriVax-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVax-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3 and 4 were observed in YF naïve subjects inoculated with either ChimeriVax-DEN2 or YF-VAX. In contrast, 100% of YF immune subjects inoculated with ChimeriVax-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2 and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that (1) the safety and immunogenicity profile of the ChimeriVax-DEN2 vaccine is consistent with that of YF-VAX, and (2) preimmunity to YF virus does not interfere with ChimeriVax-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.

Current requirements for control of live viral vaccines, including yellow fever 17D, produced from potentially neurotropic wild-type viruses include tests for neurovirulence in nonhuman primates. We have used yellow fever 17D virus as a live vector for novel flavivirus vaccines (designated ChimeriVax) against dengue, Japanese encephalitis (JE), and West Nile (WN) viruses. For control of these vaccines, it would be preferable to substitute a test in mice for the test in a higher species (monkeys). In this study, we compare the neurovirulence of ChimeriVax vaccine candidates in suckling mice inoculated by the intracerebral (IC) route with graded doses of the test article or yellow fever 17D vaccine as a reference control. Mortality ratio and survival distribution are the outcome measures. The monkey safety test is performed as described for control of yellow fever vaccines. In both mice and monkeys, all chimeric vaccines were significantly less neurovirulent than yellow fever 17D vaccine. The test in suckling mice discriminated between strains of two different vaccines (ChimeriVax-JE and ChimeriVax-DEN1) differing by a single amino acid change, and was more sensitive for detecting virulence differences than the test in monkeys. The results indicate that the suckling mouse test is simple to perform, highly sensitive and, with appropriate validation, could complement or possibly even replace the neurovirulence component of the monkey safety test. The test in infant mice is particularly useful as a means of demonstrating biological consistency across seed virus and vaccine lots.

Flaviviral diseases such as yellow fever, Japanese encephalitis (JE) and dengue hemorrhagic fever cause enormous morbidity and mortality worldwide. There is an urgent need for alternative technologies for mass vaccination against these and other diseases, particularly in the developing world. Here, we administered a live attenuated, chimeric JE vaccine (ChimeriVax)-JE) to nonhuman primates by skin microabrasion and intradermal delivery using microneedles. Both cutaneous delivery methods induced mild viremia similar in magnitude to that observed following subcutaneous (SC) injection. The duration of viremia induced by cutaneous delivery (5-7 days), however, was substantially longer than via SC (0-3 days). In addition, mean neutralizing antibody titers in cutaneous delivery groups were up to 7-fold greater than via SC injection. There were no safety issues identified and both cutaneous delivery methods appeared to be well tolerated. Thus, cutaneous delivery may represent a minimally-invasive alternative approach for flavivirus vaccines that more closely resembles the natural route of viral infection.

ChimeriVax-dengue (DEN) viruses are live attenuated vaccine candidates. They are constructed by replacing the premembrane (prM) and envelope (E) genes of the yellow fever (YF) 17D virus vaccine with the corresponding genes from wild-type DEN viruses (serotypes 1-4) isolated from humans. In this study, the growth kinetics of ChimeriVax-DEN1-4 and parent viruses (wild-type DEN-1-4 and YF 17D) were assessed in human myeloid dendritic cells (DCs) and in three hepatic cell lines (HepG2, Huh7, and THLE-3). In DC, ChimeriVax-DEN-1-4 showed similar growth kinetics to their parent viruses, wild-type DEN virus (propagated in Vero cells), or YF 17D virus (peak titers ~3-4.5 log(10) plaque-forming units (PFU)/mL at 48-72 hours post-infection). Parent wild-type DEN-1-4 viruses derived from C6/36 mosquito cells did not show any growth at a multiplicity of infection of 0.1 in DCs, except for DEN-2 virus, which grew to a modest titer of 2.5 log(10) PFU/mL at 48 hours post-infection. ChimeriVax-DEN1-4 grew to significantly lower titers (2-5 log(10) PFU/mL) than YF 17D virus in hepatic cell lines THLE-3 and HepG2, but not in Huh7 cells. These experiments suggest that ChimeriVax-DEN1-4 viruses replicate similarly to YF-VAX in DCs, but at a lower level than YF 17D virus in hepatic cell lines. The lack of growth of chimeric viruses in human hepatic cells suggests that these viruses may be less hepatotropic than YF 17D virus vaccine in humans.

St. Louis encephalitis (SLE) and West Nile (WN) flaviviruses are genetically closely related and cocirculate in the United States. Virus neutralization tests provide the most specific means for serodiagnosis of infections with these viruses. However, use of wild-type SLE and WN viral strains for laboratory testing is constrained by the biocontainment requirements. We constructed two highly attenuated yellow fever (YF) virus chimeras that contain the premembrane-envelope (prM-E) protein genes from the virulent MSI-7 (isolated in the United States) or the naturally attenuated CorAn9124 (Argentina) SLE strains. The YF/SLE (CorAn version) virus and the previously constructed YF/WN chimera were shown to specifically distinguish between confirmed human SLE and WN cases in a virus neutralization test using patient sera. These chimeras have the potential for use as diagnostic reagents and vaccines against SLE and WN.

The ability of antisera raised against a candidate Japanese encephalitis virus (JEV) vaccine, ChimeriVax-JE, and the currently licensed vaccine, JE-VAX, to protect against strains of JEV representing the four major genotypes was assessed. Neutralization assays and passive protection studies in mice showed that greatest protection was provided against strains of genotypes II and III, although some protection was also afforded against genotypes I and IV strains. ChimeriVax-JE stimulated protection that was comparable or superior to the JE-VAX control.

Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 x 10(-4) per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 x 10(-7) to 2.3 x 10(-7). Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.

The vector competence of mosquitoes for chimeric viruses being developed as vaccines to protect against dengue (DEN) virus infection were evaluated in a cooperative agreement with Acambis, Inc. Chimeric viruses have been constructed that contain the premembrane (prM) and envelope (E) genes of each of the wild-type (wt) DEN virus serotypes, DEN-1, DEN-2, DEN-3, and DEN-4, in the yellow fever (YF) vaccine virus (strain 17D) YF-VAX backbone. It was previously shown that the replication profile of ChimeriVax-DEN2 virus in Aedes albopictus C6/36 cells and in vivo in Ae. aegypti mosquitoes corresponded to that of YF-VAX virus; replication was restricted in C6/36 cells, and Ae. aegypti were poorly infected via an artificial infectious blood meal. Thus, there is very little risk of transmission by mosquitoes of ChimeriVax-DEN2 vaccine virus through the bite of a mosquito. However, because ChimeriVax-DEN 1, 2, 3, 4 viruses will be administered to humans simultaneously, growth of a mixture of ChimeriVax-DEN 1, 2, 3, 4 viruses was assessed in both C6/36 cells in culture and in the Ae. aegypti mosquito, which is the primary vector of both YF and DEN viruses. Mosquitoes were intrathoracically (IT) inoculated with virus or fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN 1, 2, 3, 4 were compared with the wt DEN and YF-VAX viruses. A quantitative real-time reverse transcriptase-polymerase chain reaction assay was developed as a method to detect and differentiate replication of each of the four ChimeriVax-DEN serotypes in the ChimeriVax-DEN 1, 2, 3, 4 tetravalent mixture. Growth of the chimeric viruses in C6/36 cells and in IT-inoculated Ae. aegypti was lower than that of YF-VAX virus; in previous studies Ae. aegypti was shown to be refractory to infection by YF-VAX virus. The growth rate of each chimeric virus was similar whether it was a single serotype infection, or part of the tetravalent mixture, and no interference by one chimeric virus over another chimeric serotype was observed. ChimeriVax-DEN viruses infected mosquitoes poorly via an infectious blood meal compared with wt DEN viruses. Therefore, it is unlikely that a mosquito feeding on a viremic vaccinee, would become infected with the chimeric viruses. Thus, there is very little potential for transmission by mosquitoes of the ChimeriVax-DEN vaccine viruses.

ChimeriVax-JE is a live, attenuated vaccine against Japanese encephalitis, using yellow fever (YF) 17D vaccine as a vector. In a double-blind phase 2 trial, 99 adults received vaccine, placebo, or YF 17D vaccine (YF-VAX). ChimeriVax-JE was well tolerated, with no differences in adverse events between treatment groups. Viremias resulting from administration of ChimeriVax-JE and YF-VAX were of short duration and low titer; 82 (94%) of 87 subjects administered graded doses (1.8-5.8 log(10)) of ChimeriVax-JE developed neutralizing antibodies. A second dose, administered 30 days later, had no booster effect. Previous inoculation with YF did not interfere with ChimeriVax-JE, but there was a suggestion (not statistically significant) that ChimeriVax-JE interfered with YF-VAX administered 30 days later. A separate study explored immunological memory both in subjects who had received ChimeriVax-JE 9 months before and in ChimeriVax-JE-naive subjects challenged with inactivated mouse-brain vaccine (JE-VAX). Anamnestic responses were observed in preimmune individuals. ChimeriVax-JE appears to be a safe vaccine that provides protective levels of neutralizing antibody after a single dose.

Yellow fever, dengue, Japanese encephalitis and tick-borne encephalitis viruses are the medically most important members of the Flavivirus genus composed primarily of arboviruses. In this paper, we review the commercially available traditional flavivirus vaccines against yellow fever, Japanese encephalitis, and tick-borne encephalitis, as well as modern approaches to flavivirus vaccines. Formalin inactivation technology has been employed to produce killed vaccines. Flaviviruses have been attenuated by multiple passages in animal tissues and cell cultures to produce empirical live attenuated vaccines. The use of traditional methods is being pursued to develop vaccines against other flavivirus diseases, such as dengue, and to improve existing vaccines, such as for Japanese encephalitis. With the recent development of infectious clones, rational approaches to attenuated flavivirus vaccines have employed the introduction of specific mutations into wild type viruses and chimerisation between different viruses. Novel methods for delivery of live vaccines, such as inoculation of infectious DNA or RNA, have been described. Other approaches, such as the construction of protein subunit, expression vector-based and naked DNA vaccines, have been proposed to create alternate vaccine candidates.

The chimeric yellow fever (YF) 17D-dengue type 2 (ChimeriVax-DEN2) vaccine virus developed by Acambis, Inc. (Cambridge, MA) contains the prM and E genes of wild-type (wt) dengue 2 (DEN-2) (strain PUO-218) virus in the YF vaccine virus (strain 17D) backbone. The potential of ChimeriVax-DEN2 virus to infect and be transmitted by Aedes aegypti, the principal DEN and YF virus mosquito vector, and Aedes albopictus, a species that occurs in areas of active transmission of YF and DEN viruses, was evaluated. Mosquitoes were intrathoracically (IT) inoculated with virus or were fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN2 were compared with the wt DEN-2 and YF 17D vaccine viruses. Replication of YF 17D virus is attenuated in cultured Ae. albopictus C6/36 mosquito cells and in Ae. aegypti and Ae. albopictus mosquitoes. Growth of ChimeriVax-DEN2 virus similarly was restricted in C6/36 cells and in mosquitoes. ChimeriVax-DEN2 replicated in 56% of IT inoculated Ae. aegypti, and virus disseminated to head tissue in 36%, with a mean viral titer of 1.8 log10 PFU/mosquito. Of mosquitoes, 16% of Ae. aegypti and 24% of Ae. albopictus were infected 14 days after a blood meal containing ChimeriVax-DEN2, but virus did not disseminate to head tissue. In contrast, DEN-2 replicated in all IT inoculated and orally infected Ae. aegypti (mean titer 5.5 log10 PFU/mosquito), and virus disseminated to head tissue in 95%. Of Ae. albopictus, 84% were infected after a blood meal containing DEN-2 virus; dissemination occurred in 36%. Replication of ChimeriVax-DEN2 virus in mosquitoes corresponded to that of YF 17D vaccine virus, which is restricted in its ability to infect and replicate in mosquitoes. Therefore, transmission of ChimeriVax-DEN2 virus by vector mosquitoes is unlikely.

A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met-->Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans.

ChimeriVax is a live, attenuated recombinant virus constructed from yellow fever (YF) 17D in which the envelope protein genes of YF 17D are replaced with the corresponding genes of another flavivirus. A ChimeriVax vaccine was developed against Japanese encephalitis (JE). A randomized, double-blind, outpatient study was conducted to compare the safety and immunogenicity of ChimeriVax-JE and YF 17D. Six YF immune and six non-immune adults were randomized to receive a single SC inoculation of ChimeriVax-JE (5log(10)PFU), ChimeriVax-JE (4log(10)PFU) or YF-VAX((R)) (5log(10)PFU). Mild, transient injection site reactions and flu-like symptoms were noted in all treatment groups, with no significant difference between the groups. Nearly all subjects inoculated with ChimeriVax-JE at both dose levels developed a transient, low-level viremia which was similar in magnitude and duration to that following YF-VAX). Neutralizing antibody seroconversion rates to ChimeriVax-JE was 100% in the high and low dose groups in both naïve and YF immune subjects; seroconversion to wild-type JE strains was similar or lower than to the homologous (vaccine) virus. Mean neutralizing antibody responses were higher in the ChimeriVax-JE high dose groups (naïve subjects LNI 1.55, PRNT(50) 254; YF immune subjects LNI 2.23, PRNT(50) 327) than in the low dose groups (naïve subjects 1.38, PRNT(50) 128; YF immune subjects LNI 1.62, PRNT(50) 270). JE antibody levels were higher in YF immune than in naïve subjects, dispelling concerns about anti-vector immunity. The safety and immunogenicity profile of ChimeriVax-JE vaccine appears to be similar to that of YF 17D. The new vaccine holds promise for prevention of JE in travelers and residents of endemic countries. The ChimeriVax technology platform is being exploited for development of new vaccines against dengue and West Nile.

Consensus sequencing of the genome of the ARILVAX live attenuated yellow fever (YF) 17D vaccine was performed directly on reconstituted virus from a vial of the vaccine secondary seed (without plaque-purification or cloning of cDNA). The genome of ARILVAX was identical in organization and size (10,862 nucleotides (nt)) to other published YF 17D sequences. A total of 12 nt heterogeneities were detected indicating that the vaccine is a heterogeneous population. Some of these indicated the presence of quasispecies with residues not reported previously for other sequenced YF 17D strains. A number of nts clearly differed from some YF vaccine strain sequences but coincided with the others, which could be due to the use of consensus sequencing approach in this study. Most (but not all) of the heterogeneities and nt differences were silent (i.e. did not result in an amino acid change). The differences are inconsequential to safety and effectiveness of ARILVAX. Other YF 17D vaccines are undoubtedly also heterogeneous and need to be re-examined using the consensus approach.

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.

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.