Evaluation of St. Louis encephalitis virus/dengue virus type 4 antigenic chimeric viruses in mice and rhesus monkeys

To develop a live attenuated virus vaccine against St. Louis encephalitis (SLE) virus, two antigenic chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue virus type 4 (DEN4) with those from SLE with or without a 30 nucleotide deletion in the DEN4 3' untranslated region of the chimeric genome. Chimeric viruses were compared with parental wild-type SLE for level of neurovirulence and neuroinvasiveness in mice and for safety, immunogenicity, and protective efficacy in rhesus monkeys. The resulting viruses, SLE/DEN4 and SLE/DEN4Delta30, had greatly reduced neuroinvasiveness in immunodeficient mice but retained neurovirulence in suckling mice. Chimerization of SLE with DEN4 resulted in only moderate restriction in replication in rhesus monkeys, whereas the presence of the Delta30 mutation led to over-attenuation. Introduction of previously described attenuating paired charge-to-alanine mutations in the DEN4 NS5 protein of SLE/DEN4 reduced neurovirulence in mice and replication in rhesus monkeys. Two modified SLE/DEN4 viruses, SLE/DEN4-436,437 clone 41 and SLE/DEN4-654,655 clone 46, have significantly reduced neurovirulence in mice and conferred protective immunity in monkeys against SLE challenge. These viruses may be considered for use as SLE vaccine candidates and for use as diagnostic reagents with reduced virulence.     

Chimeric West Nile/dengue virus vaccine candidate: preclinical evaluation in mice, geese and monkeys for safety and immunogenicity

A live attenuated virus vaccine is being developed to protect against West Nile virus (WN) disease in humans. Previously, it was found that chimeric West Nile/dengue viruses (WN/DEN4 and WN/DEN4Delta30) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue type 4 virus (DEN4) with or without a deletion of 30 nucleotides (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome were attenuated and efficacious in mice and monkeys against WN challenge. Here, we report the generation of a clinical lot of WN/DEN4Delta30 virus and its further preclinical evaluation for safety and immunogenicity in mice, geese and monkeys. The vaccine candidate had lost neuroinvasiveness in highly sensitive immunodeficient mice inoculated intraperitoneally and had greatly reduced neurovirulence in suckling mice inoculated intracerebrally (IC). Compared to the wild-type WN parent, the chimeric virus was highly restricted in replication in both murine and human neuroblastoma cells as well as in brains of suckling mice. The WN/DEN4Delta30 virus failed to infect geese, indicating that chimerization of WN with DEN4 completely attenuated WN for this avian host. This observation suggests that the WN/DEN4 chimeric viruses would be restricted in their ability to be transmitted from vaccinees to domestic or wild birds. In monkeys, the WN/DEN4Delta30 vaccine candidate was highly immunogenic despite its low level of replication with undetectable viremia. Furthermore, the WN/DEN4Delta30 vaccine virus was safe and readily induced neutralizing antibodies against WN in monkeys immune to each of the four serotypes of dengue virus. These studies confirm the attenuation of WN/DEN4Delta30 for non-human primates, including dengue-immune monkeys, and demonstrate both a highly restricted replication (>10(8)-fold decrease) in the brain of mice inoculated IC and an absence of infectivity for birds, findings that indicate this vaccine should be safe for both the recipient and the environment.     

Evaluation of the Langat/dengue 4 chimeric virus as a live attenuated tick-borne encephalitis vaccine for safety and immunogenicity in healthy adult volunteers

With the steady rise in tick-borne encephalitis virus (TBEV) infections in Europe, development of a live attenuated vaccine that will generate long-lasting immunity would be of considerable benefit. A chimeric flavivirus, designated LGT/DEN4, was previously constructed to have a genome containing the prM and E protein genes of Langat virus (LGT), a naturally attenuated member of the TBEV complex, and the remaining genetic sequences derived from dengue 4 virus (DEN4). LGT/DEN4 was highly attenuated in rodents and non-human primates, and clinical trials in humans were initiated. Twenty-eight healthy seronegative adult volunteers were randomly assigned in a 4:1 ratio to receive 10(3) plaque-forming units (PFU) of LGT/DEN4 or placebo. Volunteers were closely monitored for clinical responses and for blood chemistry and hematological changes, and the level of viremia and the magnitude and duration of the neutralizing antibody response were determined. The LGT/DEN4 vaccine was safe and viremia was seen in only one vaccinee. Infection induced a neutralizing antibody response to wild-type LGT in 80% of volunteers with a geometric mean titer (GMT) of 1:63 present on day 42 post-immunization; however the antibody response against TBEV was both much less frequent (35%) and lower in magnitude (GMT=1:9). To assess the response to a booster dose, 21 of the original 28 volunteers were re-randomized to receive a second dose of either 10(3) PFU of vaccine or placebo given 6-18 months after the first dose. The immunogenicity against either LGT or TBEV was not significantly enhanced after the second dose of vaccine. Thus, chimerization of LGT with DEN4 yielded a vaccine virus that was highly attenuated yet infectious in humans. The level of replication was sufficiently restricted to induce only a weak cross-reactive antibody response to TBEV. To provide a sufficient level of immunity to widely prevalent, highly neurovirulent strains of TBEV in humans, vaccine candidates will likely need to be based on the TBEV structural protein genes.     

Substitution of the structural genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys

Antigenic chimeric viruses in which the structural genes of dengue virus type 4 (DEN4) have been replaced with those derived from dengue virus type 2 (DEN2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. Specifically, the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions of DEN2 were substituted for the corresponding genes of wild-type rDEN4 or vaccine candidate rDEN4delta30 which contains a 30 nucleotide deletion in the 3' untranslated region. The two DEN2/4 chimeric viruses lacking the delta 30 mutation were highly attenuated in tumor-bearing SCID-HuH-7 mice, mosquitoes, and rhesus monkeys, indicating chimerization with either the CME or ME regions lead to attenuation. In mosquitoes and SCID-HuH-7 mice, addition of the delta 30 mutation to the chimeric viruses resulted in comparable or only slightly increased levels of attenuation. In rhesus monkeys, addition of the delta 30 mutation rendered the CME chimeric virus non-infectious, indicating that the attenuation resulting from chimerization and the delta 30 mutation were additive for these animals. In contrast, the attenuation in rhesus monkeys of ME chimeric virus was not significantly modified by the addition of the delta 30 mutation. The satisfactory level of attenuation and immunogenicity achieved by the ME containing DEN2/4delta 30 chimeric virus, as well as its very low infectivity for mosquitoes, make it a vaccine candidate suitable for evaluation in phase I clinical trials.     

Construction and biological properties of yellow fever 17D/dengue type 1 recombinant virus

Dengue virus, a mosquito-borne flavivirus, is one of the most formidable public health threats in tropical and subtropical regions. As yet, there is no licensed vaccine to protect against the disease. A chimeric yellow fever (YF) 17D/dengue (DEN) type 1 virus was constructed by replacing the pre-membrane and envelope genes of YF 17D virus with those from DEN 1 VeMir95 virus, a Venezuelan isolate. The chimeric YF 17D/DEN 1 VeMir95 virus was regenerated from full-length infectious clones stably propagated in Escherichia coli by transfection of Vero cells with in vitro transcribed RNA. The chimeric virus proliferated efficiently in Vero cells ( approximately 6.6 log(10) plaque-forming units/ml). The chimeric virus was not neurovirulent to 3-week-old Swiss Webster mice inoculated by the intracerebral route, in contrast to the YF 17DD vaccine strain that was lethal for 90% of the mice. The YF 17D/DEN 1 virus at Passage 6 was more attenuated for rhesus monkeys than the YF 17DD commercial vaccine after intracerebral inoculation according to the standard neurovirulence test. This virus is a potential candidate to be included in a tetravalent DEN vaccine formulation. The availability of the cloned cDNA allows further structure/function studies on the viral envelope.     

Introduction of mutations into the non-structural genes or 3' untranslated region of an attenuated dengue virus type 4 vaccine candidate further decreases replication in rhesus monkeys while retaining protective immunity

A dengue virus vaccine candidate, rDEN4Delta30, has been previously reported to be safe and immunogenic in humans, but a subset of vaccinees developed asymptomatic rash, elevation of liver enzymes and/or mild neutropenia. In the current study, mutations that had previously been shown to reduce replication of DEN4 virus in suckling mice and/or in SCID mice engrafted with human liver cells (SCID-HuH-7 mice) were introduced into rDEN4Delta30 in an attempt to further attenuate this virus. Three of the five resulting modified rDEN4Delta30 viruses showed decreased replication in SCID-HuH-7 mice relative to rDEN4Delta30. Moreover, in rhesus monkeys, two of the modified rDEN4Delta30 viruses showed a decrease in replication relative to rDEN4Delta30 while generating levels of neutralizing antibody similar to rDEN4Delta30 virus. All of the modified rDEN4Delta30 viruses completely protected immunized rhesus monkeys from challenge with wild-type DEN4 virus. Based on their attenuation for both human liver cells and rhesus monkeys, two of the modified rDEN4Delta30 vaccine candidates are currently being prepared for use in clinical trials. The application of these attenuating mutations to flavivirus vaccine development is discussed.     

Dengue virus type 3 vaccine candidates generated by introduction of deletions in the 3' untranslated region (3'-UTR) or by exchange of the DENV-3 3'-UTR with that of DENV-4

The dengue virus type 3 (DENV-3) vaccine candidate, rDEN3Delta30, was previously found to be under-attenuated in both SCID-HuH-7 mice and rhesus monkeys. Herein, two strategies have been employed to generate attenuated rDEN3 vaccine candidates which retain the full complement of structural and nonstructural proteins of DENV-3 and thus are able to induce humoral or cellular immunity to each of the DENV-3 proteins. First, using the predicted secondary structure of the 3' untranslated region (3'-UTR) of DENV-3 to design novel deletions, nine deletion mutant viruses were engineered and found to be viable. Four of nine deletion mutants replicated efficiently in Vero cells and were genetically stable. Second, chimeric rDENV-3 viruses were generated by replacement of the 3'-UTR of the rDENV-3 cDNA clone with that of rDENV-4 or rDEN4Delta30 yielding the rDEN3-3'D4 and rDEN3-3'D4Delta30 viruses, respectively. Immunization of rhesus monkeys with either of two deletion mutant viruses, rDEN3Delta30/31 and rDEN3Delta86, or with rDEN3-3'D4Delta30 resulted in infection without detectable viremia, with each virus inducing a strong neutralizing antibody response capable of conferring protection from DENV-3 challenge. The rDEN3Delta30/31 virus showed a strong host range restriction phenotype with complete loss of replication in C6/36 mosquito cells despite robust replication in Vero cells. In addition, rDEN3Delta30/31 had reduced replication in Toxorynchites mosquitoes following intrathoracic inoculation. The results are discussed in the context of vaccine development and the physical structure of the DENV 3'-UTR.     

Assurance of neuroattenuation of a live vaccine against West Nile virus: A comprehensive study of neuropathogenesis after infection with chimeric WN/DEN4Δ30 vaccine in comparison to two parental viruses and a surrogate flavivirus reference vaccine

The upsurge of West Nile virus (WNV) human infections in 2012 suggests that the US can expect periodic WNV outbreaks in the future. Availability of safe and effective vaccines against WNV in endemic areas, particularly for aging populations that are at high risk of West Nile neuroinvasive disease (WNND), could be beneficial. WN/DEN4Δ30 is a live, attenuated chimeric vaccine against WNV produced by replacement of the genes encoding the pre-membrane and envelope protein genes of the vaccine virus against dengue virus type 4 (DEN4Δ30) with corresponding sequences derived from a wild type WNV. Following intrathalamic inoculation of nonhuman primates (NHPs), a comprehensive neuropathogenesis study was performed and neurovirulence of WN/DEN4Δ30 vaccine candidate was compared to that of two parental viruses (i.e., WNV and DEN4Δ30), as well as to that of an attenuated flavivirus surrogate reference (i.e., yellow fever YF 17D). Clinical and virological data, as well as results of a semi-quantitative histopathological analysis, demonstrated that WN/DEN4Δ30 vaccine is highly attenuated for the central nervous system (CNS) of NHPs in comparison to a wild type WNV. Importantly, based on the virus replicative ability in the CNS of NHPs and the degree of induced histopathological changes, the level of neuroattenuation of WN/DEN4Δ30 vaccine was similar to that of YF 17D, and therefore within an acceptable range. In addition, we show that the DEN4Δ30 vaccine tested in this study also has a low neurovirulence profile. In summary, our results demonstrate a high level of neuroattenuation of two vaccine candidates, WN/DEN4Δ30 and DEN4Δ30. We also show here a remarkable sensitivity of our WNV-NY99 NHP model, as well as striking resemblance of the observed neuropathology to that seen in human WNND. These results support the use of this NHP model for translational studies of WNV neuropathogenesis and/or testing the effectiveness of vaccines and therapeutic approaches.     

Recombinant chimeric Japanese encephalitis virus/tick-borne encephalitis virus is attenuated and protective in mice

Tick-borne encephalitis virus (TBEV) represents one of the most dangerous human pathogens circulating in Europe and East Asia. No effective treatment for TBEV infection currently exists, and vaccination is the primary preventive measure. Although several inactivated vaccines have been licensed, the development of novel vaccines against TBEV remains a high priority in disease-endemic countries. In the present study, a live chimeric recombinant TBEV (ChinTBEV) was created by substituting the major structural genes of TBEV for the corresponding regions of Japanese encephalitis virus (JEV) live vaccine strain SA14-14-2. The resulting chimera had a small-plaque phenotype, replicated efficiently in both mammalian and mosquito cells. The preliminary data from in vitro passaging indicated the potential for stability of ChinTBEV. ChinTBEV also exhibited significantly attenuated neuroinvasiveness in mice upon either intraperitoneal or subcutaneous inoculation in comparison with its parental TBEV. Importantly, a single immunisation with ChinTBEV elicited TBEV-specific IgG and neutralising antibody responses in a dose-dependent manner, providing significant protection against lethal TBEV challenge in mice. Taken together, the results of this proof-of-concept study indicate that ChinTBEV can be further developed as a potential vaccine candidate against TBEV infection. Moreover, the construction of this type of flavivirus chimera using a JEV vaccine strain as the genetic backbone represents a universal vaccine approach.     

Generation and preclinical evaluation of a DENV-1/2 prM + E chimeric live attenuated vaccine candidate with enhanced prM cleavage

In the absence of a vaccine or sustainable vector control measures, illnesses caused by dengue virus infection remain an important public health problem in many tropical countries. During the export of dengue virus particles, furin-mediated cleavage of the prM envelope protein is usually incomplete, thus generating a mixture of immature, partially mature and mature extracellular particles. Variations in the arrangement and conformation of the envelope proteins among these particles may be associated with their different roles in shaping the antibody response. In an attempt to improve upon live, attenuated dengue vaccine approaches, a mutant chimeric virus, with enhanced prM cleavage, was generated by introducing a cleavage-enhancing substitution into a chimeric DENV-1/2 virus genome, encoding the prM + E sequence of a recent DENV-1 isolate under an attenuated DENV-2 genetic background. A modest increase in virus specific infectivity observed in the mutant chimeric virus affected neither the attenuation phenotype, when assessed in the suckling mouse neurovirulence model, nor multiplication in mosquitoes. The two chimeric viruses induced similar levels of anti-DENV-1 neutralizing antibody response in mice and rhesus macaques, but more efficient control of viremia during viral challenge was observed in macaques immunized with the mutant chimeric virus. These results indicate that the DENV-1/2 chimeric virus, with enhanced prM cleavage, could be useful as an alternative live, attenuated vaccine candidate for further tests in humans.     

Dengue-4 vaccine: neurovirulence, viraemia and immune responses in rhesus and cynomolgus monkeys

A dengue 4 (DEN-4, strain 1036-PDK 48) vaccine attenuated by passage in primary dog kidney cells was tested using rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) monkeys to determine its safety, potency, and immunogenicity. 14 rhesus monkeys were divided into 3 groups: group 1, 2 animals given control culture fluid; group 2, 2 animals given DEN-4 parental virus; group 3, 10 animals given DEN-4 vaccine virus. 10 cynomolgus were similarly grouped, but group 3 contained 6 monkeys. No significant neurovirulence was observed with parental or with DEN-4 virus passaged in primary dog kidney (PDK) cells. Both cynomolgus monkeys inoculated with DEN-4 vaccine virus developed minimal (V-1) and mild (V-2) neurovirulence-type lesions in the central nervous system, which were nondestructive in both species. All parental and vaccine viruses produced moderate to high neutralizing antibody titres. Only parental virus produced viraemia, in 2 cynomolgus monkeys. Because of its safety and avirulence in monkeys, PDK 48 is recommended for human trial.     

Immunization of rhesus monkeys with a recombinant of modified vaccinia virus Ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge

Dengue epidemics increasingly pose a public health problem in most countries of the tropical and subtropical areas. Despite decades of research, development of a safe and effective live dengue virus vaccine is still at the experimental stage. To explore an alternative vaccine strategy, we employed the highly attenuated, replication-deficient modified vaccinia Ankara (MVA) as a vector to construct recombinants for expression of the major envelope glycoprotein of one or more dengue virus serotypes. MVA recombinants expressing the highly immunogenic C-terminally truncated dengue type 2 virus (DEN2) or dengue type 4 virus (DEN4) envelope protein (E), approx. 80% of the full-length, were evaluated for their protective immunity in animal models. Each of these recombinants elicited an elevated antibody response to DEN2 or DEN4 E in mice following the booster inoculation, as detected by radio-immunoprecipitation. Recombinant MVA-DEN2 80%E, but not MVA-DEN4 80%E, induced a neutralizing antibody response. The MVA-DEN2 80%E recombinant was chosen to further evaluate its ability to induce resistance to wild type DEN2 challenge in monkeys. Monkeys immunized twice with recombinant MVA-DEN2 80%E developed a low to moderate antibody response and were partially protected against DEN2 challenge, as determined by the viremia pattern. Importantly, the subsequent study showed that all four monkeys immunized with the recombinant in a three dose schedule developed an increased level of antibodies and were completely protected against DEN2 challenge. The potential efficacy of recombinant MVA-DEN2 80%E to protect primates against dengue infection suggests that construction and evaluation of MVA recombinants expressing other serotypes of dengue virus E for use in a tetravalent vaccine strategy might be warranted.     

Construction and biological characterization of artificial recombinants between a wild type flavivirus (Kunjin) and a live chimeric flavivirus vaccine (ChimeriVax-JE)

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.     

Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells

Mutations which increase the replication of dengue viruses in cell culture would greatly facilitate the manufacture of both a live attenuated or inactivated dengue virus vaccine. We have identified eight missense mutations in dengue virus type 4 (DEN4) that increase the plaque size and kinetics of replication of recombinant DEN4 virus in Vero cells. DEN4 viruses bearing these Vero cell adaptation mutations were also evaluated for the level of replication in the brains of mice. Two of these eight recombinant viruses expressing distinct mutations in NS3 were both restricted in replication in the brains of suckling mice. In contrast, six recombinant viruses, each encoding individual mutations in NS4B (five) or in NS5 (one), were not attenuated in mouse brain. Recombinant viruses encoding various combinations of these Vero cell adaptation mutations did not demonstrate enhanced replication in Vero cells over that exhibited by the single mutations. Finally, addition of a subset of the above non-attenuating, adaptation mutations to a DEN2/4 chimeric vaccine candidate was found to increase the virus yield in Vero cells by up to 500-fold. The importance of these Vero cell adaptation mutations in flavivirus vaccine design and development is discussed.     

Attenuated West Nile viruses bearing 3'SL and envelope gene substitution mutations

Four viable West Nile (WN) 3'SL-mutant viruses were evaluated for neuroinvasiveness and neurovirulence in mice. All mutants were highly attenuated for neuroinvasiveness. However, only one of these four (WNmutE virus) was significantly attenuated for neurovirulence. To attenuate WNmutE virus further, we introduced five substitution mutations into the envelope (env) gene segment in wild-type (wt) WN and WNmutE genomes, based on differences in the env gene sequence between the live Japanese encephalitis vaccine (SA14-14-2) and its virulent parent. The env gene mutations had an attenuating effect in the context of the wt WNV genome but only a marginal enhancing effect on the attenuation of WNmutE virus.     

Induction of protective immunity against Dengue virus type 2: comparison of candidate live attenuated and recombinant vaccines

Dengue (DEN) viruses (serotypes 1 to 4) are mosquito-borne flaviviruses which cause about fifty million human infections annually and represent an expanding public health problem in the tropics. At present, there are no safe and effective vaccines which induce protective immunity to all four serotypes of DEN. Natural infection or vaccination with native and recombinant proteins may induce an immune response to the surface envelope E-protein which was shown to be protective to super-infection with homologous serotype of the virus. Purified recombinant E-protein was made in the baculovirus-Spodoptera frugiperda expression system. This protein induced neutralizing antibodies in mice. These results prompted us to immunize cynomolgus monkeys (Macaca fascicularis) with either a live attenuated DEN-2 vaccine or the recombinant E-protein complexed to aluminum hydroxide. After immunization, the monkeys were challenged with the homologous DEN virus. Serum was collected at several time points and a virus-specific antibody response including a virus neutralizing antibody response was measured. Antibody kinetics and levels were similar to those recorded in humans with a natural DEN-virus infection. Virus isolation and type specific RT-PCR were performed on the serum samples. The virus was isolated from sham vaccinated control monkeys but not from monkeys vaccinated with the live attenuated vaccine. One of the two monkeys immunized with the recombinant E-protein was also protected. Taken together these data indicate the potential of both candidate vaccines and stress the need for evaluation of different antigen presentation systems for the development of a subunit vaccine approach for DEN.     

Chimeric Sindbis/eastern equine encephalitis vaccine candidates are highly attenuated and immunogenic in mice

We developed chimeric Sindbis (SINV)/eastern equine encephalitis (EEEV) viruses and investigated their potential for use as live virus vaccines against EEEV. One vaccine candidate contained structural protein genes from a typical North American EEEV strain, while the other had structural proteins from a naturally attenuated Brazilian isolate. Both chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in mice. Vaccinated mice did not develop detectable disease or viremia, but developed high titers of neutralizing antibodies. Upon challenge with EEEV, mice vaccinated with >10(4) PFU of the chimeric viruses were completely protected from disease. These findings support the potential use of these SIN/EEEV chimeras as safe and effective vaccines.     

Pathogenicity and neurovirulence of a mutagen-attenuated Rift Valley fever vaccine in rhesus monkeys

Rhesus macaques, intravenously inoculated with virulent Rift Valley fever virus, develop viremia and biochemical evidence of liver damage and serve as a model for human disease. Some of these monkeys suffer more serious disease with hemorrhagic phenomena and approximately 20% die with frank hemorrhage. Presently, the only Rift Valley fever vaccine approved for use in humans is a formalin-killed product that requires annual booster vaccinations. Efforts to produce an improved vaccine to replace the present vaccine have led to a mutagen-attenuated strain of Rift Valley fever virus that was found to be markedly attenuated for rhesus macaques and showed promise as a vaccine candidate for human use. Neurovirulence testing in rhesus monkeys showed that, while the vaccine was not completely innocuous, residual lesions were no more severe than the currently used 17D yellow fever vaccine.     

Multiple amino acid changes at the first glycosylation motif in NS1 protein of West Nile virus are necessary for complete attenuation for mouse neuroinvasiveness

West Nile virus (WNV), like all members of the Japanese encephalitis (JE) serogroup except JE virus, contains three N-linked glycosylation (N-X-S/T) sites in the NS1 protein at asparagine residues NS1₁₃₀, NS1₁₇₅ and NS1₂₀₇. Previously we showed that the ablation of these glycosylation sites in WNV, by substitution of asparagine for alanine, attenuated mouse neuroinvasiveness; however, full attenuation was not achieved and the virus retained a neurovirulence phenotype. Sequence of viral RNA extracted from mouse brains revealed a reversion at the NS1₁₃₀ site in some mice that succumbed to the attenuated NS1_{130A/175A/207A} strain. Here, we further attenuated WNV by mutating the asparagine to serine or glutamine in addition to mutating other residues in the NS1_130-132 glycosylation motif. These mutants proved to further attenuate WNV for both neuroinvasiveness and neurovirulence in mice. NS1_{130-132QQA/175A/207A}, the most attenuated mutant virus, showed modest changes in infectivity titers versus the parental strain, was not temperature sensitive, and did not show reversion in mice. Mutant virus was completely attenuated for neuroinvasiveness after intraperitoneal inoculation with >1,000,000 PFU, and mice were protected against lethal challenge. Overall, we showed that changing the asparagine of the NS1₁₃₀ glycosylation motif to a serine or glutamine attenuated WNV further than the asparagine to alanine substitution. Further, mutating all three of the amino acids of the NS1_130-132 glycosylation motif (NTT-QQA) along with NS1₁₇₅ and NS1₂₀₇ asparagine to alanine mutations gave the most stable and attenuated strain.