Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.     

DNA vaccines against dengue virus type 2 based on truncate envelope protein or its domain III

Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2.     

A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2 as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates

The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.     

Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection‐enhancing antibody response

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection‐enhancing activities at sub‐neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM‐E) gene‐based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM‐E gene backbone showed high neutralizing antibody titers with less DENV infection‐enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.     

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.     

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.     

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.     

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.     

ChimeriVax-West Nile virus live-attenuated vaccine: preclinical evaluation of safety, immunogenicity, and efficacy

The availability of ChimeriVax vaccine technology for delivery of flavivirus protective antigens at the time West Nile (WN) virus was first detected in North America in 1999 contributed to the rapid development of the vaccine candidate against WN virus described here. ChimeriVax-Japanese encephalitis (JE), the first live- attenuated vaccine developed with this technology has successfully undergone phase I and II clinical trials. The ChimeriVax technology utilizes yellow fever virus (YF) 17D vaccine strain capsid and nonstructural genes to deliver the envelope gene of other flaviviruses as live-attenuated chimeric viruses. Amino acid sequence homology between the envelope protein (E) of JE and WN viruses facilitated targeting attenuating mutation sites to develop the WN vaccine. Here we discuss preclinical studies with the ChimeriVax-WN virus in mice and macaques. ChimeriVax-WN virus vaccine is less neurovirulent than the commercial YF 17D vaccine in mice and nonhuman primates. Attenuation of the virus is determined by the chimeric nature of the construct containing attenuating mutations in the YF 17D virus backbone and three point mutations introduced to alter residues 107, 316, and 440 in the WN virus E protein gene. The safety, immunogenicity, and efficacy of the ChimeriVax-WN(02) vaccine in the macaque model indicate the vaccine candidate is expected to be safe and immunogenic for humans.     

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.     

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.     

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.     

Early IFN-Gamma Production after YF 17D Vaccine Virus Immunization in Mice and Its Association with Adaptive Immune Responses

Yellow Fever vaccine is one of the most efficacious human vaccines ever made. The vaccine (YF 17D) virus induces polyvalent immune responses, with a mixed T_H1/T_H2 CD4⁺ cell profile, which results in robust T CD8⁺ responses and high titers of neutralizing antibody. In recent years, it has been suggested that early events after yellow fever vaccination are crucial to the development of adequate acquired immunity. We have previously shown that primary immunization of humans and monkeys with YF 17D virus vaccine resulted in the early synthesis of IFN-γ. Herein we have demonstrated, for the first time that early IFN-γ production after yellow fever vaccination is a feature also of murine infection and is much more pronounced in the C57BL/6 strain compared to the BALB/c strain. Likewise, in C57BL/6 strain, we have observed the highest CD8⁺ T cells responses as well as higher titers of neutralizing antibodies and total anti-YF IgG. Regardless of this intense IFN-γ response in mice, it was not possible to see higher titers of IgG2a in relation to IgG1 in both mice lineages. However, IgG2a titers were positively correlated to neutralizing antibodies levels, pointing to an important role of IFN-γ in eliciting high quality responses against YF 17D, therefore influencing the immunogenicity of this vaccine.     

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.     

Development,Characterization and Application of Monoclonal Antibodies against Brazilian Dengue Virus Isolates

Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV) detection through the production and characterization of 22 monoclonal antibodies (mAbs) against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3) and dengue serotype-specific (DENV-2 or -3). Additionally, some mAbs cross-reacted with yellow fever virus (YFV), West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV). None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV). Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA) for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.     

Current progress in dengue vaccines

Dengue is one of the most important emerging vector-borne viral diseases. There are four serotypes of dengue viruses (DENV), each of which is capable of causing self-limited dengue fever (DF) or even life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The major clinical manifestations of severe DENV disease are vascular leakage, thrombocytopenia, and hemorrhage, yet the detailed mechanisms are not fully resolved. Besides the direct effects of the virus, immunopathological aspects are also involved in the development of dengue symptoms. Although no licensed dengue vaccine is yet available, several vaccine candidates are under development, including live attenuated virus vaccines, live chimeric virus vaccines, inactivated virus vaccines, and live recombinant, DNA and subunit vaccines. The live attenuated virus vaccines and live chimeric virus vaccines are undergoing clinical evaluation. The other vaccine candidates have been evaluated in preclinical animal models or are being prepared for clinical trials. For the safety and efficacy of dengue vaccines, the immunopathogenic complications such as antibody-mediated enhancement and autoimmunity of dengue disease need to be considered.     

Induction of Neutralizing Antibodies against Four Serotypes of Dengue Viruses by MixBiEDIII,a Tetravalent Dengue Vaccine

The worldwide expansion of four serotypes of dengue virus (DENV) poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII) was proposed. Tandem EDIIIs of two serotypes (type 1–2 and type 3–4) of DENV connected by a Gly-Ser linker ((Gly₄Ser)₃) were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.     

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.