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.
It was found previously that induction of innate immunity, particularly chemokines, is an important mechanism of rabies virus (RABV) attenuation. To evaluate the effect of overexpression of chemokines on RABV infection, chemokines macrophage inflammatory protein 1α (MIP-1α), RANTES, and IP-10 were individually cloned into the genome of attenuated RABV strain HEP-Flury. These recombinant RABVs were characterized in vitro for growth properties and expression of chemokines. It was found that all the recombinant viruses grew as well as the parent virus, and each of the viruses expressed the intended chemokine in a dose-dependent manner. When these viruses were evaluated for pathogenicity in the mouse model, it was found that overexpression of MIP-1α further decreased RABV pathogenicity by inducing a transient innate immune response. In contrast, overexpression of RANTES or IP-10 increased RABV pathogenicity by causing neurological diseases, which is due to persistent and high-level expression of chemokines, excessive infiltration and accumulation of inflammatory cells in the central nervous system, and severe enhancement of blood-brain barrier permeability. These studies indicate that overexpression of chemokines, although important in controlling virus infection, may not always be beneficial to the host.
The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (107 PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals.
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.
Ebola virus; rabies virus; vaccine; T cell; multivalent; platform
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.
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.
Recently it was found that prior immunization with recombinant rabies virus (RABV) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) (LBNSE-GM-CSF) resulted in high innate/adaptive immune responses and protection against challenge with virulent RABV (Wen et al., JVI, 2011). In this study, the ability of LBNSE-GM-CSF to prevent animals from developing rabies was investigated in mice after infection with lethal doses of street RABV. It was found that intracerebral administration of LBNSE-GM-CSF protected more mice from developing rabies than sham-treated mice as late as day 5 after infection with street RABV. Intracerebral administration of LBNSE-GM-CSF resulted in significantly higher levels of chemokine/cytokine expression and more infiltration of inflammatory and immune cells into the central nervous system (CNS) than sham-administration or administration with UV-inactivated LBNSE-GM-CSF. Enhancement of blood-brain barrier (BBB) permeability and increases in virus neutralizing antibodies (VNA) were also observed in mice treated with LBNSE-GM-CSF. On the other hand, intracerebral administration with UV-inactivated LBNSE-GM-CSF did not increase protection despite the fact that VNA were induced in the periphery. However, intracerebral administration with chemoattractant protein-1 (MCP-1, also termed CCL2) increased significantly the protective efficacy of UV-inactivated LBNSE-GM-CSF. Together these studies confirm that direct administration of LBNSE-GM-CSF can enhance the innate and adaptive immunity as well as the BBB permeability, thus allowing infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and prevent the development of rabies.
Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90–95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection.
Our previous studies indicated that recruitment and/or activation of dendritic cells (DCs) is important in enhancing the protective immune responses against rabies virus (RABV) (L. Zhao, H. Toriumi, H. Wang, Y. Kuang, X. Guo, K. Morimoto, and Z. F. Fu, J. Virol. 84:9642-9648). To address the importance of DC activation for RABV vaccine efficacy, the genes for several DC recruitment and/or activation molecules, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein 1α (MIP-1α), were individually cloned into RABV. The ability of these recombinant viruses to activate DCs was determined in vitro and in vivo. Infection of mouse bone marrow-derived DCs with each of the recombinant viruses resulted in DC activation, as shown by increased surface expression of CD11c and CD86 as well as an increased level of alpha interferon (IFN-α) production compared to levels observed after infection with the parent virus. Intramuscular infection of mice with each of the viruses recruited and/or activated more DCs and B cells in the periphery than infection with the parent virus, leading to the production of higher levels of virus-neutralizing antibodies. Furthermore, a single immunization with recombinant RABV expressing GM-CSF or MDC protected significantly more mice against intracerebral challenge with virulent RABV than did immunization with the parental virus. Yet, these viruses did not show more virulence than the parent virus, since direct intracerebral inoculation with each virus at up to 1 × 107 fluorescent focus units each did not induce any overt clinic symptom, such as abnormal behavior, or any neurological signs. Together, these data indicate that recombinant RABVs expressing these molecules activate/recruit DCs and enhance protective immune responses.
A single intramuscular application of the live but not UV-inactivated recombinant rabies virus (RABV) variant TriGAS in mice induces the robust and sustained production of RABV-neutralizing antibodies that correlate with long-term protection against challenge with an otherwise lethal dose of the wild-type RABV. To obtain insight into the mechanism by which live TriGAS induces long-lasting protective immunity, quantitative PCR (qPCR) analysis of muscle tissue, draining lymph nodes, spleen, spinal cord, and brain at different times after TriGAS inoculation revealed the presence of significant copy numbers of RABV-specific RNA in muscle, lymph node, and to a lesser extent, spleen for several days postinfection. Notably, no significant amounts of RABV RNA were detected in brain or spinal cord at any time after TriGAS inoculation. Differential qPCR analysis revealed that the RABV-specific RNA detected in muscle is predominantly genomic RNA, whereas RABV RNA detected in draining lymph nodes is predominantly mRNA. Comparison of genomic RNA and mRNA obtained from isolated lymph node cells showed the highest mRNA-to-genomic-RNA ratios in B cells and dendritic cells (DCs), suggesting that these cells represent the major cell population that is infected in the lymph node. Since RABV RNA declined to undetectable levels by 14 days postinoculation of TriGAS, we speculate that a transient infection of DCs with TriGAS may be highly immunostimulatory through mechanisms that enhance antigen presentation. Our results support the superior efficacy and safety of TriGAS and advocate for its utility as a vaccine.
Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer.
vesicular stomatitis virus; transsynaptic infection; rabies; retrograde transneuronal tracing; in vivo; technology; polysynaptic
Our previous studies indicated that recombinant rabies viruses (rRABV) expressing chemokines or cytokines (including GM-CSF) could enhance the immunogenicity by recruiting and/or activating dendritic cells (DC). In this study, bacterial flagellin was cloned into the RABV genome and recombinant virus LBNSE-Flagellin was rescued. To compare the immunogenicity of LBNSE-Flagellin with recombinant virus expressing GMCSF (LBNSE-GMCSF), mice were immunized with each of these rRABVs by intramuscular (i.m.) or oral route. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. The i.m.-immunized mice were bled at three weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with 50 LD50 challenge virus standard (CVS-24). Orally immunized mice were boosted after three weeks and then bled and challenged one week after the booster immunization. It was found that both LBNSE-GMCSF and LBNSE-Flagellin recruited/activated more DCs and B cells in the periphery, stimulated higher levels of adaptive immune responses (VNA), and protected more mice against challenge infection than the parent virus LBNSE in both the i.m. and the orally immunized groups. Together, these studies suggest that recombinant RABV expressing GM-CSF or flagellin are more immunogenic than the parent virus in both i.m. and oral immunizations.
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R−/−) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R−/− mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R−/− mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R−/− mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving post-exposure treatment. A person, disproportionately a child, dies of rabies every 20 minutes and the cost of rabies prevention exceeds $1 billion US dollars per year. The development of a single-dose human rabies vaccine would greatly reduce the burden of rabies globally by lowering the cost associated with rabies vaccination and saving lives. Understanding how B cells develop to produce protective virus neutralizing antibodies would greatly help to achieve the goal of developing a single-dose vaccine. In this report, we show that IL-21 is critical for the induction of primary vaccine-induced anti-RABV G antibody titers and that the effects of IL-21 are highly dependent on the dose of vaccine administered. In our model of rabies immunogenicity and protection, the lack of IL-21 receptor influenced the detection of B cells in germinal centers in lymph nodes or of plasma cells in bone marrow after immunization with low or high doses of vaccine, respectively. Overall, these preliminary results indicate that IL-21 has the potential to influence B cell development and functions in the context of rabies vaccine-induced immunity and protection.
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.
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.
Previously, we showed that overexpression of MIP-1α in mouse brain further decreased rabies virus (RABV) pathogenicity (L. Zhao, H. Toriumi, Y. Kuang, H. Chen, and Z. F. Fu, J. Virol., 83:11808-11818, 2009). In the present study, the immunogenicity of recombinant RABV expressing MIP-1α (rHEP-MIP1α) was determined. It was found that intramuscular immunization of BALB/c mice with rHEP-MIP1α resulted in a higher level of expression of MIP-1α at the site of inoculation, increased recruitment of dendritic cells (DCs) and mature B cells into the draining lymph nodes and the peripheral blood, and higher virus-neutralizing antibody titers than immunization with the parent rHEP and recombinant RABVs expressing RANTES (CCL5) or IP-10 (CXCL10). Our data thus demonstrate that expression of MIP-1α not only reduces viral pathogenicity but also enhances immunogenicity by recruiting DCs and B cells to the site of immunization, the lymph nodes, and the blood.
The neurotropic rabies virus (RABV) has developed several evasive strategies, including immunoevasion, to successfully infect the nervous system (NS) and trigger a fatal encephalomyelitis. Here we show that expression of LGP2, a protein known as either a positive or negative regulator of the RIG-I-mediated innate immune response, is restricted in the NS. We used a new transgenic mouse model (LGP2 TG) overexpressing LGP2 to impair the innate immune response to RABV and thus revealed the role of the RIG-I-mediated innate immune response in RABV pathogenesis. After infection, LGP2 TG mice exhibited reduced expression of inflammatory/chemoattractive molecules, beta interferon (IFN-β), and IFN-stimulated genes in their NS compared to wild-type (WT) mice, demonstrating the inhibitory function of LGP2 in the innate immune response to RABV. Surprisingly, LGP2 TG mice showed more viral clearance in the brain and lower morbidity than WT mice, indicating that the host innate immune response, paradoxically, favors RABV neuroinvasiveness and morbidity. LGP2 TG mice exhibited similar neutralizing antibodies and microglia activation to those of WT mice but showed a reduction of infiltrating CD4+ T cells and less disappearance of infiltrating CD8+ T cells. This occurred concomitantly with reduced neural expression of the IFN-inducible protein B7-H1, an immunoevasive protein involved in the elimination of infiltrated CD8+ T cells. Our study shows that the host innate immune response favors the infiltration of T cells and, at the same time, promotes CD8+ T cell elimination. Thus, to a certain extent, RABV exploits the innate immune response to develop its immunoevasive strategy.
Rabies virus (RABV) can infect many different species of warm-blooded animals. Glycoprotein G plays a key role in viral pathogenicity and neurotropism, and includes antigenic domains that are responsible for membrane fusion and host cell receptor recognition.
A case of buffalo rabies in China was diagnosed by direct fluorescent antibody test, G gene reverse-transcriptase polymerase chain reaction, and RABV mouse inoculation test. Molecular characterization of the RABV was performed using DNA sequencing, phylogenetic analysis and amino acid sequence comparison based on the G gene from different species of animals.
The results confirmed that the buffalo with suspected rabies was infected by RABV, which was genetically closely related to HNC (FJ602451) that was isolated from cattle in China in 2007. Comparison of the G gene among different species of animal showed that there were almost no amino acid changes among RABVs isolated from the same species of animals that distributed in a near region. However, there were many changes among RABVs that were isolated from different species of animal, or the same species from different geographic regions. This is believed to be the first case report of buffalo rabies in China, and the results may provide further information to understand the mechanism by which RABV breaks through the species barrier.
Induction of innate immunity, particularly through the induction of interferon and chemokines, by rabies virus (RABV) infection has been reported to be inversely correlated with pathogenicity. To further investigate the association between the expression of chemokines and RABV infection, laboratory-attenuated RABV (B2C) and wild-type (wt) RABV (DRV) were administered to Balb/c mice intramuscularly. Chemokine expression, inflammatory cell infiltration, and blood-brain barrier (BBB) permeability were evaluated at various time points after infection. At day 3 post infection (p.i.) there was very little inflammation in the central nervous system (CNS) and BBB permeability did not change in mice infected with either virus when compared with mock-infected mice. At 6 day p.i., infection with B2C induced the expression of inflammatory chemokines and infiltration of inflammatory cells into the CNS, while these changes were minimal in DRV-infected mice. Furthermore, infection with B2C significantly enhanced BBB permeability comparing to infection with DRV. Among the upregulated chemokines, the expression of IP-10 was best correlated with infiltration of inflammatory cells into the CNS and enhancement of BBB permeability. These data indicate that laboratory-attenuated RABV induces expression of chemokines and infiltration of inflammatory cells into the CNS. Upregulation of chemokines by B2C may have triggered the change in BBB permeability, which helps infiltration of inflammatory cells into the CNS, and thus attenuation of RABV.
rabies; innate immunity
Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD50) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 106 PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 108 PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 108 PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.
Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.
Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.
Viruses are obligate parasites. The progression of their life cycle depends on their hijacking the cellular metabolism and machinery. Human neurons produce TLR3, a protein involved in early host defence mechanisms and the modulation of neuronal survival. Rabies virus is a neurotropic virus, infecting mainly neurons. In this study, we showed that rabies virus exploits TLR3 function to store viral proteins and viral genomic material in particular areas of the cell where virus multiplication occurs. We found that, during the course of infection, large (1–3 µm) spherical inclusions were formed within the region around the nucleus. These inclusions were composed of an inner core of aggregated TLR3 surrounded by a coat of viral proteins and genomic material. These inclusions were revealed to be the previously described Negri Bodies (NBs). In absence of TLR3, NBs were no longer formed and virus multiplication rate decreased. Mice deficient in TLR3 were more resistant to rabies and had lower levels of infection in their brains. This study shows how neurotropic viruses, such as rabies virus, hijack normal functions of neuronal proteins and use cell compartmentalisation to promote viral multiplication.
Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.
vaccine; anthrax; rhabdovirus; rabies; glycoprotein
Rabies virus (RABV) causes a fatal infection of the central nervous systems (CNS) of warm-blooded animals. Once the clinical symptoms develop, rabies is almost invariably fatal. The mechanism of RABV pathogenesis remains poorly understood. Recent studies have shown that microRNA (miRNA) plays an important role in the pathogenesis of viral infections. Our recent findings have revealed that infection with laboratory-fixed rabies virus strain can induce modulation of the microRNA profile of mouse brains. However, no previous report has evaluated the miRNA expression profile of mouse brains infected with RABV street strain.
The results of microarray analysis show that miRNA expression becomes modulated in the brains of mice infected with street RABV. Quantitative real-time PCR assay of the differentially expressed miRNAs confirmed the results of microarray assay. Functional analysis showed the differentially expressed miRNAs to be involved in many immune-related signaling pathways, such as the Jak-STAT signaling pathway, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The predicted expression levels of the target genes of these modulated miRNAs were found to be correlated with gene expression as measured by DNA microarray and qRT-PCR.
RABV causes significant changes in the miRNA expression profiles of infected mouse brains. Predicted target genes of the differentially expression miRNAs are associated with host immune response, which may provide important information for investigation of RABV pathogenesis and therapeutic method.
Street strain rabies virus; Brain infection; MicroRNA profiling; Gene profiling; Target prediction; Functional enrichment
The rabies virus (RABV) is highly neurotropic and it uses evasive strategies to successfully evade the host immune system. Because rabies is often fatal, understanding the basic processes of the virus-host interactions, particularly in the initial events of infection, is critical for the design of new therapeutic approaches to target RABV. Here, we examined the possible role of dendritic cells (DCs) in the transmission of RABV to neural cells at peripheral site of exposure. Viral replication only occurred at a low level in the DC cell line, JAWS II, after its infection with either pathogenic RABV (CVS strain) or low-pathogenic RABV (ERA strain), and no progeny viruses were produced in the culture supernatants. However, both viral genomic RNAs were retained in the long term after infection and maintained their infectivity. The biggest difference between CVS and ERA was in their ability to induce type I interferons. Although the ERA-infected JAWS II cells exhibited cytopathic effect and were apparently killed by normal spleen cells in vitro, the CVS-infected JAWS II cells showed milder cytopathic effect and less lysis when cocultured with spleen cells. Strongly increased expression of major histocompatibility complex classes I, costimulatory molecules (CD80 and CD86), type I interferons and Toll- like receptor 3, and was observed only in the ERA-inoculated JAWS II cells and not in those inoculated with CVS. During the silencing of the cellular immune response in the DCs, the pathogenic CVS strain cryptically maintained an infectious viral genome and was capable of transmitting infectious RABV to permissive neural cells. These findings demonstrate that DCs may play a role in the passive carriage of RABV during natural rabies infections.
Rabies virus; Dendritic cells; Immune invasion
Rabies virus (RABV) causes severe neurological disease and death. As an important mechanism for generating genetic diversity in viruses, homologous recombination can lead to the emergence of novel virus strains with increased virulence and changed host tropism. However, it is still unclear whether recombination plays a role in the evolution of RABV. In this study, we isolated and sequenced four circulating RABV strains in China. Phylogenetic analyses identified a novel lineage of hybrid origin that comprises two different strains, J and CQ92. Analyses revealed that the virus 3′ untranslated region (UTR) and part of the N gene (approximate 500 nt in length) were likely derived from Chinese lineage I while the other part of the genomic sequence was homologous to Chinese lineage II. Our findings reveal that homologous recombination can occur naturally in the field and shape the genetic structure of RABV populations.
Three anti-rabies virus (RABV) nucleoprotein (N) monoclonal antibodies (Mab) were characterized by immunofluorescence assays, western blotting, and immunohistochemistry. One of these Mabs recognized the antigen by all of the assays, while the other two recognized N only in the native form in the immunofluorescence assay. These data, together with epitope mapping studies, suggest that two anti-N Mabs recognize conformational epitopes located within the N-terminal region of the RABV N protein. The availability of Mabs specific for both linear and epitope-specific antibodies should prove valuable for rabies diagnosis as well as for RABV N protein structure–function studies.