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1.  Preclinical Development of Inactivated Rabies Virus–Based Polyvalent Vaccine Against Rabies and Filoviruses 
The Journal of Infectious Diseases  2015;212(Suppl 2):S414-S424.
We previously described the generation of a novel Ebola virus (EBOV) vaccine based on inactivated rabies virus (RABV) containing EBOV glycoprotein (GP) incorporated in the RABV virion. Our results demonstrated safety, immunogenicity, and protective efficacy in mice and nonhuman primates (NHPs). Protection against viral challenge depended largely on the quality of the humoral immune response against EBOV GP.
Here we present the extension and improvement of this vaccine by increasing the amount of GP incorporation into virions via GP codon-optimization as well as the addition of Sudan virus (SUDV) and Marburg virus (MARV) GP containing virions. Immunogenicity studies in mice indicate similar immune responses for both SUDV GP and MARV GP compared to EBOV GP. Immunizing mice with multiple antigens resulted in immune responses similar to immunization with a single antigen. Moreover, immunization of NHP with the new inactivated RABV EBOV vaccine resulted in high titer neutralizing antibody levels and 100% protection against lethal EBOV challenge when applied with adjuvant.
Our results indicate that an inactivated polyvalent vaccine against RABV filoviruses is achievable. Finally, the novel vaccines are produced on approved VERO cells and a clinical grade RABV/EBOV vaccine for human trials has been produced.
PMCID: PMC4564550  PMID: 26063224
Ebola virus; Sudan virus; Marburg virus; rabies virus; vaccine; filovirus; polyvalent vaccine
2.  Rhabdovirus-Based Vaccine Platforms against Henipaviruses 
Journal of Virology  2014;89(1):144-154.
The emerging zoonotic pathogens Hendra virus (HeV) and Nipah virus (NiV) are in the genus Henipavirus in the family Paramyxoviridae. HeV and NiV infections can be highly fatal to humans and livestock. The goal of this study was to develop candidate vaccines against henipaviruses utilizing two well-established rhabdoviral vaccine vector platforms, recombinant rabies virus (RABV) and recombinant vesicular stomatitis virus (VSV), expressing either the codon-optimized or the wild-type (wt) HeV glycoprotein (G) gene. The RABV vector expressing the codon-optimized HeV G showed a 2- to 3-fold increase in incorporation compared to the RABV vector expressing wt HeV G. There was no significant difference in HeV G incorporation in the VSV vectors expressing either wt or codon-optimized HeV G. Mice inoculated intranasally with any of these live recombinant viruses showed no signs of disease, including weight loss, indicating that HeV G expression and incorporation did not increase the neurotropism of the vaccine vectors. To test the immunogenicity of the vaccine candidates, we immunized mice intramuscularly with either one dose of the live vaccines or 3 doses of 10 μg chemically inactivated viral particles. Increased codon-optimized HeV G incorporation into RABV virions resulted in higher antibody titers against HeV G compared to inactivated RABV virions expressing wt HeV G. The live VSV vectors induced more HeV G-specific antibodies as well as higher levels of HeV neutralizing antibodies than the RABV vectors. In the case of killed particles, HeV neutralizing serum titers were very similar between the two platforms. These results indicated that killed RABV with codon-optimized HeV G should be the vector of choice as a dual vaccine in areas where rabies is endemic.
IMPORTANCE Scientists have been tracking two new viruses carried by the Pteropid fruit bats: Hendra virus (HeV) and Nipah virus (NiV). Both viruses can be fatal to humans and also pose a serious risk to domestic animals. A recent escalation in the frequency of outbreaks has increased the need for a vaccine that prevents HeV and NiV infections. In this study, we performed an extensive comparison of live and killed particles of two recombinant rhabdoviral vectors, rabies virus and vesicular stomatitis virus (VSV), expressing wild-type or codon-optimized HeV glycoprotein, with the goal of developing a candidate vaccine against HeV. Based on our data from the presented mouse immunogenicity studies, we conclude that a killed RABV vaccine would be highly effective against HeV infections and would make an excellent vaccine candidate in areas where both RABV and henipaviruses pose a threat to human health.
PMCID: PMC4301098  PMID: 25320306
3.  Rabies Virus Is Recognized by the NLRP3 Inflammasome and Activates Interleukin-1β Release in Murine Dendritic Cells 
Journal of Virology  2013;87(10):5848-5857.
Inflammasome activation is important for the development of an effective host defense against many pathogens, including RNA viruses. However, the mechanism by which the inflammasome recognizes RNA viruses and its role in rabies virus (RABV) pathogenicity and immunogenicity remain poorly defined. To determine the function of the inflammasome in response to RABV infection, we infected murine bone marrow-derived dendritic cells (BMDCs) with RABV. Our results indicate that the infection of BMDCs with RABV induces both the production of pro-interleukin-1β (pro-IL-1β) and its processing, resulting in the secretion of active IL-1β through activation of the NLRP3-, ASC-, and caspase-1-dependent inflammasome. As previously shown for the induction of type I interferon by RABV, the induction of pro-IL-1β also depends upon IPS-1. We demonstrate that both the production of pro-IL-1β and activation of the inflammasome require viral replication. We also demonstrate that increased viral replication in BMDCs derived from IFNAR-deficient mice resulted in significantly more IL-1β release. Additionally, IL-1 receptor-deficient mice show an increase in RABV pathogenicity. Taken together, these results indicate an important role of the inflammasome in innate immune recognition of RABV.
PMCID: PMC3648142  PMID: 23487464
4.  Reverse genetics of Mononegavirales: How they work, new vaccines, and new cancer therapeutics 
Virology  2015;0:331-344.
The order Mononegavirales includes five families: Bornaviridae, Filoviridae, Nyamaviridae, Paramyxoviridae, and Rhabdoviridae. The genome of these viruses is one molecule of negative-sense single strand RNA coding for five to ten genes in a conserved order. The RNA is not infectious until packaged by the nucleocapsid protein and transcribed by the polymerase and co-factors. Reverse genetics approaches have answered fundamental questions about the biology of Mononegavirales. The lack of icosahedral symmetry and modular organization in the genome of these viruses has facilitated engineering of viruses expressing fluorescent proteins, and these fluorescent proteins have provided important insights about the molecular and cellular basis of tissue tropism and pathogenesis. Studies have assessed the relevance for virulence of different receptors and the interactions with cellular proteins governing the innate immune responses. Research has also analyzed the mechanisms of attenuation. Based on these findings, ongoing clinical trials are exploring new live attenuated vaccines and the use of viruses re-engineered as cancer therapeutics.
PMCID: PMC4557643  PMID: 25702088
Mononegavirales; Reverse genetics; Tropism; Attenuation; Pathology; Receptors; Tropism; Innate immunity; Vaccines; Cancer therapy
5.  Dendritic cells infected by recombinant rabies virus vaccine vector expressing HIV-1 Gag are immunogenic even in presence of vector-specific immunity 
Vaccine  2010;29(1):130-140.
Dendritic cells (DC) are the most potent antigen presenting cells whose ability to interact with T cells, B cells and NK cells has led to their extensive use in vaccine design. Here, we designed a DC-based HIV-1 vaccine using an attenuated rabies virus vector expressing HIV-1 Gag (RIDC-Gag). To test this, BALB/c mice were immunized with RIDC-Gag, and the primary, secondary as well as humoral immune responses were monitored. Our results indicate that RIDC-Gag stimulated HIV-1 Gag-specific immune responses in mice. When challenged with vaccinia virus (VV) expressing HIV-1 Gag, they elicited a potent Gag-specific recall response characterized by CD8+ T cells expressing multiple cytokines that were capable of specifically lysing Gag-pulsed target cells. Moreover, RIDC-Gag also enhanced CD8+ T cell responses via a homologous prime-boost regimen. These results show that a DC-based vaccine using live RV is immunogenic and a potential candidate for a therapeutic HIV-1 vaccine.
PMCID: PMC2997164  PMID: 20728525
6.  Rabies virus-based vaccines elicit neutralizing antibodies, poly-functional CD8+ T cell, and protect rhesus macaques from AIDS-like disease after SIVmac251 challenge 
Vaccine  2009;28(2):299-308.
Highly attenuated rabies virus (RV) vaccine vectors were evaluated for their ability to protect against highly pathogenic SIVmac251 challenge. Mamu-A*01 negative rhesus macaques were immunized in groups of four with either: RV expressing SIVmac239-GagPol, a combination of RV expressing SIVmac239-Env and RV expressing SIVmac239-GagPol, or with empty RV vectors. Eight weeks later animals received a booster immunization with a heterologous RV expressing the same antigens. At twelve weeks post-boost, all animals were challenged intravenously with 100 TCID50 of pathogenic SIVmac251-CX. Immunized macaques in both vaccine groups had 1.3–1.6-log fold decrease in viral set point compared to control animals. The GagPol/Env immunized animals also had a significantly lower peak viral load. When compared to control animals following challenge, vaccinated macaques had a more rapid induction of SIVmac251 neutralizing antibodies and of CD8+ T cell responses to various SIV epitopes. Moreover, vaccinated macaques better-maintained peripheral memory CD4+ T cells and were able to mount a poly-functional CD8+ T cell response in the mucosa. These findings indicate promise for RV-based vectors and have important implications for the development of an efficacious HIV vaccine.
PMCID: PMC2826816  PMID: 19879223
rabies virus; HIV-1; vaccine; SIVmac251; rhabdovirus
7.  Interferon-β expressed by a rabies virus-based HIV-1 vaccine vector serves as a molecular adjuvant and decreases pathogenicity 
Virology  2008;382(2):226-238.
Type I interferon is important in anti-viral responses and in coordinating the innate immune response. Here we explore the use of interferon-β to adjuvant the response to a rabies virus (RV) vaccine vector expressing both HIV-1 Gag and IFN-β. Viral load and immune responses of immunized mice were analyzed over time. Our results indicate that the RV expressing IFN-β (IFN(+)) is highly attenuated when compared to control RV and demonstrate that the expression of IFN-β reduces viral replication approximately 100-fold. Despite the decrease in replication, those mice immunized with the IFN(+) RV had a significantly greater number of activated CD8+ T cells. The increased activation of CD8+ T cells was dependent on IFN-β signaling, as we saw no difference following infection of IFNAR−/− mice. Although mice immunized with IFN(+) have a greater primary immune response than controls, immunized mice that were challenged with vaccinia expressing Gag had no significant difference in the number or functionality of CD8+ T cells. The increased CD8+ T cell activation in the presence of IFN-β, even with greatly reduced viral replication, indicates the beneficial effect of IFN-β for the host.
PMCID: PMC2645003  PMID: 18945463
Vaccine; Adjuvant; Interferon-beta; Rabies
8.  Safety and Serological Response to a Matrix Gene-deleted Rabies Virus-based Vaccine Vector in Dogs 
Vaccine  2014;32(15):1716-1719.
Dogs account for the majority of human exposures and deaths due to rabies virus (RABV) worldwide. In this report, we show that a replication-deficient RABV-based vaccine in which the matrix gene is deleted (RABV- M) is safe and induces rapid and potent VNA titers after a single inoculation in dogs. Average VNA titers peaked at 3.02 or 5.11 International Units (IU/ml) by 14 days post-immunization with a single dose of 106 or 107 focus forming units (ffu), respectively, of RABV- M. By day 70 post immunization, all dogs immunized with either dose of vaccine showed VNA titers >0.5 IU/ml, the level indicative of a satisfactory immunization. Importantly, no systemic or local reactions were noted in any dog immunized with RABV- M. The elimination of dog rabies through mass vaccination is hindered by limited resources, requirement for repeat vaccinations often for the life of a dog, and in some parts of the world, inferior vaccine quality. Our preliminary safety and immunogenicity data in dogs suggest that RABV- M might complement currently used inactivated RABV-based vaccines in vaccination campaigns by helping to obtain 100% response in vaccinated dogs, thereby increasing overall vaccination coverage.
PMCID: PMC3966478  PMID: 24508037
canine; rabies; replication-deficient RABV; vaccine
9.  A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies 
The Journal of general virology  2005;86(Pt 5):1435-1440.
Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.
PMCID: PMC1361274  PMID: 15831955
10.  Small-Molecule Probes Targeting the Viral PPxY-Host Nedd4 Interface Block Egress of a Broad Range of RNA Viruses 
Journal of Virology  2014;88(13):7294-7306.
Budding of filoviruses, arenaviruses, and rhabdoviruses is facilitated by subversion of host proteins, such as Nedd4 E3 ubiquitin ligase, by viral PPxY late (L) budding domains expressed within the matrix proteins of these RNA viruses. As L domains are important for budding and are highly conserved in a wide array of RNA viruses, they represent potential broad-spectrum targets for the development of antiviral drugs. To identify potential competitive blockers, we used the known Nedd4 WW domain-PPxY interaction interface as the basis of an in silico screen. Using PPxY-dependent budding of Marburg (MARV) VP40 virus-like particles (VLPs) as our model system, we identified small-molecule hit 1 that inhibited Nedd4-PPxY interaction and PPxY-dependent budding. This lead candidate was subsequently improved with additional structure-activity relationship (SAR) analog testing which enhanced antibudding activity into the nanomolar range. Current lead compounds 4 and 5 exhibit on-target effects by specifically blocking the MARV VP40 PPxY-host Nedd4 interaction and subsequent PPxY-dependent egress of MARV VP40 VLPs. In addition, lead compounds 4 and 5 exhibited antibudding activity against Ebola and Lassa fever VLPs, as well as vesicular stomatitis and rabies viruses (VSV and RABV, respectively). These data provide target validation and suggest that inhibition of the PPxY-Nedd4 interaction can serve as the basis for the development of a novel class of broad-spectrum, host-oriented antivirals targeting viruses that depend on a functional PPxY L domain for efficient egress.
IMPORTANCE There is an urgent and unmet need for the development of safe and effective therapeutics against biodefense and high-priority pathogens, including filoviruses (Ebola and Marburg) and arenaviruses (e.g., Lassa and Junin) which cause severe hemorrhagic fever syndromes with high mortality rates. We along with others have established that efficient budding of filoviruses, arenaviruses, and other viruses is critically dependent on the subversion of host proteins. As disruption of virus budding would prevent virus dissemination, identification of small-molecule compounds that block these critical viral-host interactions should effectively block disease progression and transmission. Our findings provide validation for targeting these virus-host interactions as we have identified lead inhibitors with broad-spectrum antiviral activity. In addition, such inhibitors might prove useful for newly emerging RNA viruses for which no therapeutics would be available.
PMCID: PMC4054416  PMID: 24741084
11.  Recombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo 
Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus–based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of β-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.
PMCID: PMC4362357  PMID: 26015984
12.  A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence 
Virology  2012;434(1):18-26.
We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RVΔG-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RVΔG-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RVΔG-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVΔG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.
PMCID: PMC3484205  PMID: 22889613
Ebola virus; rabies virus; vaccine; neurovirulence; mice
13.  Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein 
Vaccine  2012;30(43):6136-6141.
We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing ebolavirus (EBOV) glycoprotein (GP) that induce humoral immunity against each virus and confer protection from both lethal RABV and mouse-adapted EBOV challenge in mice. Here, we expand our investigation of the immunogenic properties of these bivalent vaccines in mice. Both live and killed vaccines induced primary EBOV GP-specific T-cells and a robust recall response as measured by interferon-γ ELISPOT assay. In addition to cellular immunity, an effective filovirus vaccine will likely require a multivalent humoral immune response against multiple virus species. As a proof-of-principle experiment, we demonstrated that inactivated RV-GP could be formulated with another inactivated RABV vaccine expressing the nontoxic fragment of botulinum neurotoxin A heavy chain (HC50) without a reduction in immunity to each component. Finally, we demonstrated that humoral immunity to GP could be induced by immunization of mice with inactivated RV-GP in the presence of pre-existing immunity to RABV. The ability of these novel vaccines to induce strong humoral and cellular immunity indicates that they should be further evaluated in additional animal models of infection.
PMCID: PMC3434297  PMID: 22884661
Ebola virus; rabies virus; vaccine; T cell; multivalent; platform
15.  Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses 
PLoS ONE  2013;8(6):e67123.
This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8+ T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8+ T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8+ T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8+ T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.
PMCID: PMC3694142  PMID: 23840600
16.  Antibody Quality and Protection from Lethal Ebola Virus Challenge in Nonhuman Primates Immunized with Rabies Virus Based Bivalent Vaccine 
PLoS Pathogens  2013;9(5):e1003389.
We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.
Author Summary
Ebola virus (EBOV) has been associated with outbreaks in human and nonhuman primate populations since 1976. With a fatality rate approaching 90%, EBOV is one of the most lethal infectious diseases in humans. The increased frequency of EBOV outbreaks along with its potential to be used as a bioterrorism agent has dramatically strengthened filovirus vaccine research and development. While there are currently no approved vaccines or post exposure treatments available for human use, several vaccine candidates have shown to protect nonhuman primates from lethal EBOV challenge. Our primary focus is to develop vaccine candidates to protect humans and endangered wildlife species at risk of infection in Africa. Here, we evaluated the efficacy and immunogenicity of our dual vaccines against EBOV and rabies virus (RABV) in rhesus macaques. Our live replication-competent vaccine provided 100% protection following EBOV challenge while the replication-deficient and inactivated candidates provided 50% protection. Interestingly, protection is dependent on the quality of the antibodies rather than the quantity. All three RABV-based EBOV vaccines did induce antibody levels necessary for protection from RABV infection. These results encourage the further development of these novel dual vaccines directed against two of the most lethal viral diseases.
PMCID: PMC3667758  PMID: 23737747
17.  Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein 
The Journal of General Virology  2013;94(Pt 2):284-292.
European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). However, the determinants of viral fitness and pathogenicity are poorly understood. Full-length genome clones based on the highly attenuated, non-neuroinvasive, RABV vaccine strain (SAD-B19) were constructed with the glycoprotein (G) of either SAD-B19 (SN), of EBLV-1 (SN-1) or EBLV-2 (SN-2). In vitro characterization of SN-1 and SN-2 in comparison to wild-type EBLVs demonstrated that the substitution of G affected the final virus titre and antigenicity. In vivo, following peripheral infection with a high viral dose (104 f.f.u.), animals infected with SN-1 had reduced survivorship relative to infection with SN, resulting in survivorship similar to animals infected with EBLV-1. The histopathological changes and antigen distribution observed for SN-1 were more representative of those observed with SN than with EBLV-1. EBLV-2 was unable to achieve a titre equivalent to that of the other viruses. Therefore, a reduced-dose experiment (103 f.f.u.) was undertaken in vivo to compare EBLV-2 and SN-2, which resulted in 100 % survivorship for all recombinant viruses (SN, SN-1 and SN-2) while clinical disease developed in mice infected with the EBLVs. These data indicate that interspecies replacement of G has an effect on virus titre in vitro, probably as a result of suboptimal G–matrix protein interactions, and influences the survival outcome following a peripheral challenge with a high virus titre in mice.
PMCID: PMC3709617  PMID: 23100360
18.  Immune Clearance of Attenuated Rabies Virus Results in Neuronal Survival with Altered Gene Expression 
PLoS Pathogens  2012;8(10):e1002971.
Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent “mark” on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these “cured” neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that can persist or progress in the absence of sustained viral antigen.
Author Summary
Rabies is an ancient and fatal neurological disease of animals and humans, caused by infection of the central nervous system (CNS) with Rabies virus (RABV). It is estimated that nearly 55,000 human RABV fatalities occur each year, though this number is likely much higher due to unreported exposures or failure of diagnosis. No treatment has been identified to cure disease after onset of symptoms. Neurovirologists still do not know the cause of rabies' dramatic symptoms and fatality, though it is thought to be due to neuronal loss or dysfunction. Here, we use a novel approach to permanently and genetically tag infected cells so that they can be identified after the infection has been cleared. This allowed us to define neuronal survival time following infection, and to assess neuronal function through gene expression analysis. We found that RABV infection does not lead to loss of neurons, but rather induces a permanent change in gene expression that may be related to the ability of RABV to cause permanent CNS disease. Our study provides evidence that viral infection of the brain can initiate long-term changes that may have consequences for nervous system health, even after the virus has been cleared from the CNS.
PMCID: PMC3469654  PMID: 23071441
19.  Replication-Deficient Rabies Virus–Based Vaccines Are Safe and Immunogenic in Mice and Nonhuman Primates 
The Journal of Infectious Diseases  2009;200(8):1251-1260.
Although current postexposure prophylaxis rabies virus (RV) vaccines are effective, ~40,000–70,000 rabies-related deaths are reported annually worldwide. The development of effective formulations requiring only 1–2 applications would significantly reduce mortality. We assessed in mice and nonhuman primates the efficacy of replication-deficient RV vaccine vectors that lack either the matrix (M) or phosphoprotein (P) gene. A single dose of M gene–deficient RV induced a more rapid and efficient anti-RV response than did P gene–deficient RV immunization. Furthermore, the M gene–deleted RV vaccine induced 4-fold higher virus-neutralizing antibody (VNA) levels in rhesus macaques than did a commercial vaccine within 10 days after inoculation, and at 180 days after immunization rhesus macaques remained healthy and had higher-avidity antibodies, higher VNA titers, and a more potent antibody response typical of a type 1 T helper response than did animals immunized with a commercial vaccine. The data presented in this article suggest that the M gene–deleted RV vaccine is safe and effective and holds the potential of replacing current pre- and postexposure RV vaccines.
PMCID: PMC3388800  PMID: 19764884
20.  Immunization of mice with the non-toxic HC50 domain of botulinum neurotoxin presented by rabies virus particles induces a strong immune response affording protection against high-dose botulinum neurotoxin challenge 
Vaccine  2011;29(28):4638-4645.
We previously showed that rabies virus (RABV) virions are excellent vehicles for antigen presentation. Here, a reverse genetic approach was applied to generate recombinant RABV that express a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin type A (BoNT/A) and RABV glycoprotein (G). To promote surface expression and incorporation of HC50/A into RABV virions, the RABV glycoprotein (G) ER translocation sequence, various fragments of RABV ectodomain (ED) and cytoplasmic domain were fused to HC50/A. The HC50/A chimeric proteins were expressed on the surface of cells infected with all of the recombinant RABVs, however, the highest level of surface expression was detected by utilizing 30 amino acids of the RABV G ED (HV50/A-E30). Our results also indicated that this chimeric protein was effectively incorporated into RABV virions. Immunization of mice with inactivated RABV-HC50/A-E30 virions induced a robust anti-HC50/A IgG antibody response that efficiently neutralized circulating BoNT/A in vivo, and protected mice against 1000 fold the lethal dose of BoNT/A.
PMCID: PMC3114282  PMID: 21549784
21.  Inactivated or Live-Attenuated Bivalent Vaccines That Confer Protection against Rabies and Ebola Viruses ▿  
Journal of Virology  2011;85(20):10605-10616.
The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.
PMCID: PMC3187516  PMID: 21849459
22.  Rabies Virus (RV) Glycoprotein Expression Levels Are Not Critical for Pathogenicity of RV▿  
Journal of Virology  2010;85(2):697-704.
Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain.
PMCID: PMC3020019  PMID: 21068252
Journal of immunological methods  2009;353(1-2):78-86.
Using Strepavidin as a scaffold, we have assembled a composite immunotoxin that consists of recombinant Pseudomonas exotoxin A subunit (PE38) and recombinant 25-D1.16 Fab fragment which recognizes the SIINFEKL (pOV8) peptide from ovalbumin in association with H-2Kb MHC class I protein. The composite immunotoxin exercises cytotoxicity against H-2Kb+ cells sensitized with pOV8 peptide but not with irrelevant peptide. Specific binding of the immunotoxin to H-2Kb+ cells infected with recombinant rabies virus (RV) expressing pOV8 epitope (RV-pOV8) resulted in the suppression of the production of virus particles by the infected cells. This strategy allows readily produce different immunotoxins with desired specificity by combining various targeting and toxin molecules. The results provide a proof of concept that composite immunotoxins can be utilized as novel immunotherapeutics to stop virus spread in the acute phase of the infection allowing winning time for the development of protective immune response.
PMCID: PMC2823984  PMID: 19932697
TCR-like antibody; peptide-MHC ligands; Pseudomonas exotoxin; composite immunotoxin; rabies virus
24.  Induction of Neutralizing Antibody Responses to Anthrax Protective Antigen by Using Influenza Virus Vectors: Implications for Disparate Immune System Priming Pathways▿  
Journal of Virology  2010;84(16):8300-8307.
Viral vectors based on influenza virus, rabies virus (RV), and vaccinia virus (VV) were used to express large polypeptide segments derived from the Bacillus anthracis protective antigen (PA). For the infectious influenza virus vector and recombinant VV constructs, the receptor binding domain (RBD or domain 4) or the lethal and edema factor binding domain (LEF or domain 1′) were engineered into functional chimeric hemagglutinin (HA) glycoproteins. In the case of the RV vector, the viral glycoprotein (G) was used as a carrier for RBD in an inactivated form of the vector. These constructs were examined by using multiple homologous and heterologous prime/boost immunization regimens in order to optimize the induction of α-PA antibody responses. Several immunization combinations were shown to induce high titers of antibody recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an in vitro anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties.
PMCID: PMC2916528  PMID: 20504926
25.  Characterization of a Single-Cycle Rabies Virus-Based Vaccine Vector ▿  
Journal of Virology  2010;84(6):2820-2831.
Recombinant rabies virus (RV)-based vectors have demonstrated their efficacy in generating long-term, antigen-specific immune responses in murine and monkey models. However, replication-competent viral vectors pose significant safety concerns due to vector pathogenicity. RV pathogenicity is largely attributed to its glycoprotein (RV-G), which facilitates the attachment and entry of RV into host cells. We have developed a live, single-cycle RV by deletion of the G gene from an RV vaccine vector expressing HIV-1 Gag (SPBN-ΔG-Gag). Passage of SPBN-ΔG-Gag on cells stably expressing RV-G allowed efficient propagation of the G-deleted RV. The in vivo immunogenicity data comparing single-cycle RV to a replication-competent control (BNSP-Gag) showed lower RV-specific antibodies; however, the overall isotype profiles (IgG2a/IgG1) were similar for the two vaccine vectors. Despite this difference, mice immunized with SPBN-ΔG-Gag and BNSP-Gag mounted similar levels of Gag-specific CD8+ T-cell responses as measured by major histocompatibility complex class I Gag-tetramer staining, gamma interferon-enzyme-linked immunospot assay, and cytotoxic T-cell assay. Moreover, these cellular responses were maintained equally at immunization titers as low as 103 focus-forming units for both RV vaccine vectors. CD8+ T-cell responses were significantly enhanced by a boost with a single-cycle RV complemented with a heterologous vesicular stomatitis virus glycoprotein. These findings demonstrate that single-cycle RV is an effective alternative to replication-competent RV vectors for future development of vaccines for HIV-1 and other infectious diseases.
PMCID: PMC2826042  PMID: 20053743

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