Growth characteristics of the veterinary vaccine candidate ChimeriVax-West Nile (WN) virus in Aedes and Culex mosquitoes

In 1999 West Nile (WN) virus was introduced to North America where this flavivirus has spread rapidly among wildlife (especially birds) transmitted by various species of mosquitoes (Diptera: Culicidae). Increasing numbers of cases and deaths among humans, horses and other domestic animals require development of effective vaccines. 'ChimeriVax-West Nile(vet)' is being developed for use as a veterinary vaccine to protect against WN infection. This chimeric virus contains the pre-membrane (prM) and envelope (E) genes from the wild-type WN NY99 virus (isolated from a flamingo in New York zoo during the 1999 WN epidemic) in the backbone of yellow fever (YF) 17D vaccine virus. Replication kinetics of ChimeriVax-WN(vet) virus were evaluated in mosquito cell culture (Aedes albopictus C6/36), in WN vector mosquitoes [Culex tritaeniorhynchus Giles, Cx. nigripalpus Theobald and Cx. quinquefasciatus Say (Diptera: Culicidae)] and in YF vectors [Aedes aegypti (L) and Ae. albopictus (Skuse)], to determine whether these mosquitoes become infected through feeding on a viraemic vaccine, and their potential infectivity to transmit the virus. Growth of ChimeriVax-WN(vet) virus was found to be restricted in mosquitoes, compared to WN virus in Ae. albopictus C6/36 cells. When inoculated intrathoracically, ChimeriVax-WN(vet) and YF 17D viruses did not replicate in Cx. tritaeniorhynchus or Cx. nigripalpus; replication was very restricted compared to the wild-type WN virus in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. When fed on hanging drops with ChimeriVax-WN(vet) virus (7.7 log10 PFU/mL), none of the Culex mosquitoes became infected; one Ae. albopictus and 10% of the Ae. aegypti became infected, but the titre was very low and virus did not disseminate to head tissue. ChimeriVax-WN(vet) virus had a replication profile similar to that of the attenuated vaccine virus YF 17D, which is not transmitted by mosquitoes. These results suggest that the natural mosquito vectors of WN and YF viruses, which may incidentally take a bloodmeal from a vaccinated host, will not become infected with ChimeriVax-WN(vet) virus.     

Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.     

Safety testing for neurovirulence of novel live, attenuated flavivirus vaccines: infant mice provide an accurate surrogate for the test in monkeys.

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.     

Exchanging the yellow fever virus envelope proteins with Modoc virus prM and E proteins results in a chimeric virus that is neuroinvasive in SCID mice

A chimeric flavivirus infectious cDNA was constructed by exchanging the premembrane (prM) and envelope (E) genes of the yellow fever virus vaccine strain 17D (YF17D) with the corresponding genes of Modoc virus (MOD). This latter virus belongs to the cluster of the "not-known vector" flaviviruses and is, unlike YF17D, neuroinvasive in SCID mice. Replication of in vitro-transcribed RNA from this chimeric flavivirus was shown by [(3)H]uridine labeling and RNA analysis. Expression of the MOD prM and E proteins was monitored by radioimmunoprecipitation and revealed that the MOD proteins were correctly and efficiently produced from the chimeric precursor protein. The MOD E protein was shown to be N-linked glycosylated, whereas prM, as predicted from the genome sequence, did not contain N-linked carbohydrates. In Vero cells, the chimeric virus replicated with a similar efficiency as the parental viruses, although it formed smaller plaques than YF17D and MOD. In SCID mice that had been infected intraperitoneally with the chimeric virus, the viral load increased steadily until death. The MOD/YF virus, like MOD from which it had acquired the prM and E structural proteins, but unlike YF, proved neuroinvasive in SCID mice. Animals developed neurological symptoms about 15 days after inoculation and died shortly thereafter. The distribution of MOD/YF RNA in the brain of infected mice was similar to that observed in MOD-infected mice. The observations provide compelling evidence that the determinants of neuroinvasiveness of flaviviruses are entirely located in the envelope proteins prM and E.     

Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus

Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers. Two D2/WN chimeras (D2/WN-E2 and -V2) engineered in the backbone of the PDK53-E and -V viruses retained all of the PDK-53 vaccine characteristic phenotypic markers of attenuation and were immunogenic in mice and protected mice from a high-dose 10(7) PFU challenge with wild-type WN NY99 virus. This report further supports application of the genetic background of the D2 PDK-53 virus as a carrier for development of live-attenuated, chimeric flavivirus vaccines in general and the development of a chimeric D2/WN vaccine virus against WN disease in particular.     

Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines

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.     

Comparative neuropathogenesis and neurovirulence of attenuated flaviviruses in nonhuman primates

Based on previous preclinical evaluation in mice and monkeys, the chimeric TBEV/DEN4Delta30 virus, carrying the prM and E protein genes from a highly virulent Far Eastern strain of tick-borne encephalitis virus (TBEV) on the backbone of a nonneuroinvasive dengue type 4 virus (DEN4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by TBEV. However, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to be evaluated. In the present study, we compared the neuropathogeneses of the chimeric TBEV/DEN4Delta30 virus; Langat virus (LGTV), a former live TBEV vaccine; and yellow fever 17D virus vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. TBEV/DEN4Delta30 and YF 17D demonstrated remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in the central nervous system (CNS) that were high in cerebral hemispheres but progressively decreased toward the spinal cord. In contrast, the neurovirulence of LGTV exhibited the reverse profile, progressing from the site of inoculation toward the cerebellum and spinal cord. Analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism associated with all three attenuated viruses. Nevertheless, TBEV/DEN4Delta30 virus exhibited higher neurovirulence in monkeys than either LGTV or YF 17D, suggesting insufficient attenuation. These results provide insight into the neuropathogenesis associated with attenuated flaviviruses that may guide the design of safe vaccines.     

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.     

The Synergistic Effect of Combined Immunization with a DNA Vaccine and Chimeric Yellow Fever/Dengue Virus Leads to Strong Protection against Dengue

The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.     

Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization,immunogenicity, and protection against dengue encephalitis in the mouse model

Two yellow fever virus (YFV)/dengue virus chimeras which encode the prM and E proteins of either dengue virus serotype 2 (dengue-2 virus) or dengue-4 virus within the genome of the YFV 17D strain (YF5.2iv infectious clone) were constructed and characterized for their properties in cell culture and as experimental vaccines in mice. The prM and E proteins appeared to be properly processed and glycosylated, and in plaque reduction neutralization tests and other assays of antigenic specificity, the E proteins exhibited profiles which resembled those of the homologous dengue virus serotypes. Both chimeric viruses replicated in cell lines of vertebrate and mosquito origin to levels comparable to those of homologous dengue viruses but less efficiently than the YF5.2iv parent. YFV/dengue-4 virus, but not YFV/dengue-2 virus, was neurovirulent for 3-week-old mice by intracerebral inoculation; however, both viruses were attenuated when administered by the intraperitoneal route in mice of that age. Single-dose inoculation of either chimeric virus at a dose of 10(5) PFU by the intraperitoneal route induced detectable levels of neutralizing antibodies against the homologous dengue virus strains. Mice which had been immunized in this manner were fully protected from challenge with homologous neurovirulent dengue viruses by intracerebral inoculation compared to unimmunized mice. Protection was associated with significant increases in geometric mean titers of neutralizing antibody compared to those for unimmunized mice. These data indicate that YFV/dengue virus chimeras elicit antibodies which represent protective memory responses in the mouse model of dengue encephalitis. The levels of neurovirulence and immunogenicity of the chimeric viruses in mice correlate with the degree of adaptation of the dengue virus strain to mice. This study supports ongoing investigations concerning the use of this technology for development of a live attenuated viral vaccine against dengue viruses.     

Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response

We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against DEN-2 virus challenge, indicating that protection was mediated by the DEN-2 virus prM- and E-specific immune responses. YF/DEN vaccine-primed CD8 T cells expanded and were efficiently recruited into the central nervous systems of DEN-2 virus challenged mice. At 5 days after challenge, 3 to 4% of CD8 T cells in the spleen were specific for the prM and E proteins, and 34% of CD8 T cells in the central nervous system recognized these proteins. Depletion of either CD4 or CD8 T cells, or both, strongly reduced the protective efficacy of the YF/DEN virus, stressing the key role of the antiviral T-cell response.     

Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.     

Neuroadapted yellow fever virus 17D: genetic and biological characterization of a highly mouse-neurovirulent virus and its infectious molecular clone

A neuroadapted strain of yellow fever virus (YFV) 17D derived from a multiply mouse brain-passaged virus (Porterfield YF17D) was additionally passaged in SCID and normal mice. The virulence properties of this virus (SPYF) could be distinguished from nonneuroadapted virus (YF5.2iv, 17D infectious clone) by decreased average survival time in SCID mice after peripheral inoculation, decreased average survival time in normal adult mice after intracerebral inoculation, and occurrence of neuroinvasiveness in normal mice. SPYF exhibited more efficient growth in peripheral tissues of SCID mice than YF5.2iv, resulting in a more rapid accumulation of virus burden, but with low-titer viremia, at the time of fatal encephalitis. In cell culture, SPYF was less efficient in replication than YF5.2iv in all cell lines tested. The complete nucleotide sequence of SPYF revealed 29 nucleotide substitutions relative to YF5.2iv, and these were distributed throughout the genome. There were a total of 13 predicted amino acid substitutions, some of which correspond to known differences among the Asibi, French viscerotropic virus, French neurotropic vaccine, and YF17D vaccine strains. The envelope (E) protein contained five substitutions, within all three functional domains. Substitutions were also present in regions encoding the NS1, NS2A, NS4A, and NS5 proteins and in the 3' untranslated region (UTR). Construction of YFV harboring all of the identified coding nucleotide substitutions and those in the 3' UTR yielded a virus whose cell culture and pathogenic properties, particularly neurovirulence and neuroinvasiveness for SCID mice, generally resembled those of the original SPYF isolate. These findings implicate the E protein and possibly other regions of the genome as virulence determinants during pathogenesis of neuroadapted YF17D virus in mice. The determinants affect replication efficiency in both neural and extraneural tissues of the mouse and confer some limited host-range differences in cultured cells of nonmurine origin.     

Molecular analysis of yellow fever virus 17DD vaccine strain.

The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared worldwide. As part of a broader approach to determine the genetic variability in YF 17D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purified directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF 17D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot hybridization of virion RNAs of purified 17DD with two other strains of YF 17D virus revealed only genome-sized molecules for all three viruses. These observations suggest that the vaccine phenotype is primarily associated with the accumulation of mutations.     

Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development

Attenuation markers of the candidate dengue 2 (D2) PDK-53 vaccine virus are encoded by mutations that reside outside of the structural gene region of the genome. We engineered nine dengue virus chimeras containing the premembrane (prM) and envelope (E) genes of wild-type D1 16007, D3 16562, or D4 1036 virus within the genetic backgrounds of wild-type D2 16681 virus and the two genetic variants (PDK53-E and PDK53-V) of the D2 PDK-53 vaccine virus. Expression of the heterologous prM-E genes in the genetic backgrounds of the two D2 PDK-53 variants, but not that of wild-type D2 16681 virus, resulted in chimeric viruses that retained PDK-53 characteristic phenotypic markers of attenuation, including small plaque size and temperature sensitivity in LLC-MK(2) cells, limited replication in C6/36 cells, and lack of neurovirulence in newborn ICR mice. Chimeric D2/1, D2/3, and D2/4 viruses replicated efficiently in Vero cells and were immunogenic in AG129 mice. Chimeric D2/1 viruses protected adult AG129 mice against lethal D1 virus challenge. Two tetravalent virus formulations, comprised of either PDK53-E- or PDK53-V-vectored viruses, elicited neutralizing antibody titers in mice against all four dengue serotypes. These antibody titers were similar to the titers elicited by monovalent immunizations, suggesting that viral interference did not occur in recipients of the tetravalent formulations. The results of this study demonstrate that the unique attenuation loci of D2 PDK-53 virus make it an attractive vector for the development of live attenuated flavivirus vaccines.     

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.     

Expression of foreign protein epitopes at the surface of recombinant yellow fever 17D viruses based on three-dimensional modeling of its envelope protein

The yellow fever (YF) 17D vaccine is a live attenuated virus, and its genetic manipulation constitutes a new platform for vaccine development. In this article we review one of the possible approaches to enable this development, which is the insertion of foreign protein epitopes into different locations of the genome. We describe the three-dimensional (3D) modeling of the YF 17D virus E protein structure based on tick-borne encephalitis (TBE) and the identification of a potential insertion site located at the YF 17D fg loop. Further 3D analysis revealed that it is possible to accommodate inserts of different sizes and amino acid composition in the flavivirus E protein fg loop. We demonstrate that seven YF 17D viruses bearing foreign epitopes that vary in sequence and length show differential growth characteristics in cell culture. The testing of recombinant viruses for mouse neurovirulence suggests that insertions at the 17D E protein fg loop do not compromise the attenuated phenotypes of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines.     

Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever

Background

Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable.

Methodology/Principal Findings

A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10⁵ TCID₅₀. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles.

Conclusions/Significance

The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.     

A DNA Vaccine against Yellow Fever Virus: Development and Evaluation

Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.