Intramuscular inoculation of mice with the live-attenuated recombinant rabies virus TriGAS results in a transient infection of the draining lymph nodes and a robust, long-lasting protective immune response against rabies.

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.

Postexposure treatment with the live-attenuated rabies virus (RV) vaccine TriGAS triggers the clearance of wild-type RV from the Central Nervous System (CNS) through the rapid induction of genes relevant to adaptive immunity in CNS tissues.

Postexposure treatment (PET) of wild-type rabies virus (RV)-infected mice with a live-attenuated triple-glycoprotein RV variant (TriGAS) promotes survival but does not prevent the pathogenic RV from invading and replicating in the brain. Successful PET is associated with the induction of a robust virus-neutralizing antibody response and clearance of the wild-type RV from brain tissues. Comparison of the transcriptomes of normal mouse brain with those of wild-type-RV-infected mice that had received either mock or TriGAS PET treatment revealed that many of the host genes activated in the mock-treated mice represent type I interferon (IFN) response genes. This indicates that RV infection induces an early type I IFN response that is unable to control the infection. In contrast, most of the activated genes in the brain of the RV-infected, TriGAS-treated mouse play a role in adaptive immunity, including the regulation of T cell activation, T cell differentiation, and the regulation of lymphocyte and mononuclear cell proliferation. These findings were confirmed by quantitative PCR (qPCR) array studies, which showed that 3 genes in particular, encoding chemokine ligand 3 (Ccl3), natural killer cell activator 2 (interleukin 12B [IL-12B]), and granzyme A (GzmA), were activated earlier and to a greater extent in the brains of RV-infected mice treated with TriGAS than in the brains of mock-treated mice. The activation of these genes, known to play key roles in the regulation of lymphocyte and mononuclear cell proliferation, is likely an important part of the mechanism by which TriGAS mediates its PET activity.

Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus.

Rabies remains an important public health problem with more than 95% of all human rabies cases caused by exposure to rabid dogs in areas where effective, inexpensive vaccines are unavailable. Because of their ability to induce strong innate and adaptive immune responses capable of clearing the infection from the CNS after a single immunization, live-attenuated rabies virus (RV) vaccines could be particularly useful not only for the global eradication of canine rabies but also for late-stage rabies postexposure prophylaxis of humans. To overcome concerns regarding the safety of live-attenuated RV vaccines, we developed the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS. In contrast to most attenuated recombinant RVs generated thus far, SPBAANGAS-GAS-GAS is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or have inherited deficits in immune function (e.g., antibody production or type I IFN signaling), as well as normal adult animals. In addition, SPBAANGAS-GAS-GAS induces immune mechanisms capable of containing a CNS infection with pathogenic RV, thereby preventing lethal rabies encephalopathy. The lack of pathogenicity together with excellent immunogenicity and the capacity to deliver immune effectors to CNS tissues makes SPBAANGAS-GAS-GAS a promising vaccine candidate for both the preexposure and postexposure prophylaxis of rabies.

Intravenous inoculation of a bat-associated rabies virus causes lethal encephalopathy in mice through invasion of the brain via neurosecretory hypothalamic fibers.

The majority of rabies virus (RV) infections are caused by bites or scratches from rabid carnivores or bats. Usually, RV utilizes the retrograde transport within the neuronal network to spread from the infection site to the central nervous system (CNS) where it replicates in neuronal somata and infects other neurons via trans-synaptic spread. We speculate that in addition to the neuronal transport of the virus, hematogenous spread from the site of infection directly to the brain after accidental spill over into the vascular system might represent an alternative way for RV to invade the CNS. So far, it is unknown whether hematogenous spread has any relevance in RV pathogenesis. To determine whether certain RV variants might have the capacity to invade the CNS from the periphery via hematogenous spread, we infected mice either intramuscularly (i.m.) or intravenously (i.v.) with the dog-associated RV DOG4 or the silver-haired bat-associated RV SB. In addition to monitoring the progression of clinical signs of rabies we used immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to follow the spread of the virus from the infection site to the brain. In contrast to i.m. infection where both variants caused a lethal encephalopathy, only i.v. infection with SB resulted in the development of a lethal infection. While qRT-PCR did not reveal major differences in virus loads in spinal cord or brain at different times after i.m. or i.v. infection of SB, immunohistochemical analysis showed that only i.v. administered SB directly infected the forebrain. The earliest affected regions were those hypothalamic nuclei, which are connected by neurosecretory fibers to the circumventricular organs neurohypophysis and median eminence. Our data suggest that hematogenous spread of SB can lead to a fatal encephalopathy through direct retrograde invasion of the CNS at the neurovascular interface of the hypothalamus-hypophysis system. This alternative mode of virus spread has implications for the post exposure prophylaxis of rabies, particularly with silver-haired bat-associated RV.

The glycoprotein and the matrix protein of rabies virus affect pathogenicity by regulating viral replication and facilitating cell-to-cell spread.

While the glycoprotein (G) of rabies virus (RV) is known to play a predominant role in the pathogenesis of rabies, the function of the RV matrix protein (M) in RV pathogenicity is not completely clear. To further investigate the roles of these proteins in viral pathogenicity, we constructed chimeric recombinant viruses by exchanging the G and M genes of the attenuated SN strain with those of the highly pathogenic SB strain. Infection of mice with these chimeric viruses revealed a significant increase in the pathogenicity of the SN strain bearing the RV G from the pathogenic SB strain. Moreover, the pathogenicity was further increased when both G and M from SB were introduced into SN. Interestingly, the replacement of the G or M gene or both in SN by the corresponding genes of SB was associated with a significant decrease in the rate of viral replication and viral RNA synthesis. In addition, a chimeric SN virus bearing both the M and G genes from SB exhibited more efficient cell-to-cell spread than a chimeric SN virus in which only the G gene was replaced. Together, these data indicate that both G and M play an important role in RV pathogenesis by regulating virus replication and facilitating cell-to-cell spread.

Special Issue: Rabies Symptoms, Diagnosis, Prophylaxis, and Treatment.

Rabies is an acute, progressive, incurable viral encephalitis found throughout the world. Despite being one of the oldest recognized pathogens, its impact remains substantial in public health, veterinary medicine, and conservation biology.[...].

Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine.

Lagos bat virus (LBV) is a phylogroup II lyssavirus exclusively found in Africa. Previous studies indicated that this virus is lethal to mice after intracranial and intramuscular inoculation. The antigenic composition of LBV differs substantially from that of rabies virus (RABV) and current rabies vaccines do not provide cross protection against phylogroup II lyssaviruses. To investigate the potential role of the LBV matrix protein (M) and glycoprotein (G) in pathogenesis, reverse genetics technology was used to construct recombinant viruses. The genes encoding the glycoprotein, or the matrix and glycoprotein of the attenuated RABV strain SPBN, were replaced with those of LBV resulting in SPBN-LBVG and SPBN-LBVM-LBVG, respectively. To evaluate the immunogenicity of the LBV G, the recombinant RABV SPBNGAS-LBVG-GAS was constructed with the LBV G inserted between two mutated RABV G genes (termed GAS). All the recombinant viruses were lethal to mice after intracranial (i.c.) inoculation although the pathogenicity of SPBNGAS-LBVG-GAS was lower compared to the other recombinant viruses. Following intramuscular (i.m.) inoculation, only SPBN-LBVM-LBVG was lethal to mice, indicating that both the M and G of LBV play a role in the pathogenesis. Most interestingly, serum collected from mice that were inoculated i.m. with SPBNGAS-LBVG-GAS neutralized phylogroup I and II lyssaviruses including RABV, Duvenhage virus (DUVV), LBV, and Mokola virus (MOKV), indicating that this recombinant virus has potential to be developed as a pan-lyssavirus vaccine.

Vaccination of small Asian mongoose (Herpestes javanicus) against rabies.

Oral vaccination of free-ranging wildlife is a promising technique in rabies control. The small Asian mongoose (Herpestes javanicus) is an important reservoir of rabies on several Caribbean islands, but no vaccines have been evaluated for this species. Captive mongooses were used to test the safety and efficacy of the commercially licensed vaccinia-rabies glycoprotein (V-RG) recombinant vaccine and a newly developed genetically engineered oral rabies virus vaccine (SPBNGA-S). In one study using V-RG, no vaccinated animals developed detectable rabies virus-neutralizing antibodies, and all but one died after experimental challenge with rabies virus. In contrast, all animals given SPBNGA-S demonstrated seroconversion within 7 to 14 days after vaccination and survived rabies virus challenge. On the basis of these preliminary results indicating the greater efficacy of SPBNGA-S vs. V-RG vaccine, additional investigations will be necessary to determine the optimal dose and duration of vaccination, as well as incorporation of the SPBNGA-S vaccine into edible bait.

Oral vaccination of dogs with recombinant rabies virus vaccines.

Oral rabies virus (RV) vaccines are used to immunize a diversity of mammalian carnivores, but no single biological is effective for all major species. Recently, advances in reverse genetics have allowed the design of recombinant RV for consideration as new vaccines. The objective of this experiment was to examine the safety, immunogenicity and efficacy of recombinant RV vaccines administered to captive dogs by the oral route, compared to a commercial vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine. Animals consisted of naive purpose-bred beagles of both sexes, and were 6 months of age or older. Dogs were randomly assigned to one of six groups, and received either diluent or vaccine (PBS; V-RG; RV SN10-333; RV SPBN-Cyto c; RV SPBNGA; RV SPBNGAGA), with at least six animals per group. On day 0, 1 ml of each vaccine (or PBS) was administered to the oral cavity of each dog, at an approximate concentration of 10(8) to 10(9) TCID50. After vaccination, dogs were observed daily and bled weekly, for 5 weeks, prior to RV challenge. No signs of illness related to vaccination were detected during the observation period. Excluding the controls, RV neutralizing antibodies were detected in the majority of animals within 1-2 weeks of primary vaccination. Thereafter, all dogs were inoculated in the masseter muscle with a street virus of canine origin. All control animals developed rabies, but no vaccinates succumbed, with the exception of a single dog in the V-RG group. Review of these preliminary data demonstrates the non-inferiority of recombinant RV products, as concerns both safety and efficacy, and supports the suggestion that these vaccines may hold promise for future development as oral immunogens for important carnivore species, such as dogs.

Management of rabies in humans.

Rabies is a fatal disease in humans, and, to date, the only survivors of the disease have received rabies vaccine before the onset of illness. The approach to management of the rabies normally should be palliative. In unusual circumstances, a decision may be made to use an aggressive approach to therapy for patients who present at an early stage of clinical disease. No single therapeutic agent is likely to be effective, but a combination of specific therapies could be considered, including rabies vaccine, rabies immunoglobulin, monoclonal antibodies, ribavirin, interferon-alpha, and ketamine. Corticosteroids should not be used. As research advances, new agents may become available in the future for the treatment of human rabies.

Recombinant rhabdoviruses as potential vaccines for HIV-1 and other diseases.

The failure to develop vaccines to protect against important infectious diseases such as human immunodeficiency virus type I (HIV-1) or Hepatitis C virus (HCV) has increased the interest in new vaccine strategies. One of these methods is immunization with an attenuated recombinant viral vector expressing a foreign antigen, which could protect individuals from later exposure to the respective pathogen. A new method to recover a non-segmented negative-stranded RNA virus (NNSV) from cDNA was described for the first time for rabies virus (RV), a member of the rhabdovirus family. The same approach was successfully used for another rhabdovirus, vesicular stomatitis virus (VSV), and opened the possibility to use rhabdoviruses as vaccine vehicles and biomedical tools. Further research showed that the genomes of rhabdoviruses are highly flexible, easy to manipulate, and able to express large and even multiple foreign genes, and therefore are excellent vaccine candidates. In addition, it has been shown for both RV and VSV that their single surface glycoprotein G, which is responsible for attachment and fusion to the host cell, can functionally be replaced by other viral or cellular glycoproteins. This review gives an overview of the use of RV and VSV as promising new candidates in the fight against HIV-1 and other human diseases.

New approaches to the prevention and eradication of rabies.

Despite significant progress in improving the pre- and postexposure prophylaxis of human rabies, the development of better and more cost-effective vaccines and antiviral therapeutics remains a major goal for the treatment of human rabies, the control of animal rabies and particularly for the eradication of rabies virus reservoirs in terrestrial wildlife. In this review, we discuss the structural requirements for an effective rabies vaccine, as well as new strategies currently in use for the development of safer and more potent rabies vaccines for rabies prophylaxis and eradication. Finally, we discuss new immune therapeutics aimed at replacing the conventional administration of antirabies immunoglobulin used in rabies post-exposure prophylaxis in humans.

Glycoprotein of nonpathogenic rabies viruses is a major inducer of apoptosis in human jurkat T cells.

This study sought to identify the RV protein that causes apoptosis. For this purpose, we first compared the ability of G and N proteins of a pathogenic and a nonpathogenic strain to trigger apoptosis of Jurkat rtTA by using an inducible Tet-on expression system. Then we analyzed apoptosis induced by a reverse genetic-engineered recombinant rabies virus in which the G gene from a nonpathogenic strain was replaced by its pathogenic strain counterpart. No other virus proteins than G of nonpathogenic RV strains induce apoptosis, and the G polypeptide of RV is a critical determinant for apoptosis in human cells.

Neuropeptide Y up-regulation in cerebrocortical neurons after Borna Disease Virus infection is unrelated to brain inflammation in rats.

Neuropeptides participate in the pathophysiology of cerebral inflammatory diseases. We analyzed the involvement of neuropeptide Y (NPY) in rat brain infected with Borna Disease Virus (BDV). NPY expressing cerebrocortical neurons were increased during the acute stage of BDV-induced encephalitis. The increase was resistant to immunosuppression by systemic dexamethasone, which greatly reduced inflammatory reactions in the brain. This indicates that the increase of cerebrocortical NPY expression is not causally related to inflammation. As cerebral NPY is known to be increased during experimental seizures and to have anticonvulsive actions, we propose that NPY up-regulated during BDV encephalitis limits seizures known to be associated with Borna Disease.

Limited brain metabolism changes differentiate between the progression and clearance of rabies virus.

Central nervous system (CNS) metabolic profiles were examined from rabies virus (RABV)-infected mice that were either mock-treated or received post-exposure treatment (PET) with a single dose of the live recombinant RABV vaccine TriGAS. CNS tissue harvested from mock-treated mice at middle and late stage infection revealed numerous changes in energy metabolites, neurotransmitters and stress hormones that correlated with replication levels of viral RNA. Although the large majority of these metabolic changes were completely absent in the brains of TriGAS-treated mice most likely due to the strong reduction in virus spread, TriGAS treatment resulted in the up-regulation of the expression of carnitine and several acylcarnitines, suggesting that these compounds are neuroprotective. The most striking change seen in mock-treated RABV-infected mice was a dramatic increase in brain and serum corticosterone levels, with the later becoming elevated before clinical signs or loss of body weight occurred. We speculate that the rise in corticosterone is part of a strategy of RABV to block the induction of immune responses that would otherwise interfere with its spread. In support of this concept, we show that pharmacological intervention to inhibit corticosterone biosynthesis, in the absence of vaccine treatment, significantly reduces the pathogenicity of RABV. Our results suggest that widespread metabolic changes, including hypothalamic-pituitary-adrenal axis activation, contribute to the pathogenesis of RABV and that preventing these alterations early in infection with PET or pharmacological blockade helps protect brain homeostasis, thereby reducing disease mortality.

The role of toll-like receptors in the induction of immune responses during rabies virus infection.

The host response to infection generally begins with interactions between pathogen-associated molecular patterns common to a variety of infectious agents and reciprocal pattern-recognition receptors (PRRs) expressed by cells of the innate immune system. The innate responses triggered by these interactions contribute to the early, innate control of infection as well as the induction of pathogen-specific adaptive immunity. The outcome of infection with wild-type rabies virus is particularly dependent upon the rapid induction of innate and adaptive immune mechanisms that can prevent the virus from reaching central nervous system (CNS) tissues, where it can evade immune clearance. However, laboratory strains that reach the CNS can be cleared, and this has evidently occurred in individuals with rabies. Therefore, PRRs may be active in the periphery and the CNS during rabies virus infection, possibly depending upon the nature of the infecting virus. To investigate these possibilities, we first examined the outcome of infection with attenuated rabies virus in mice lacking MyD88, an adaptor protein that is used to activate the transcription factor NF-κB by a number of PRRs including all of the Toll-like receptors (TLRs) except for TLR3. Finding that attenuated rabies virus mediates lethal disease in the absence of MyD88, we then examined the effects of the deletion of receptors using MyD88 including TLRs 2, 4, 7, and 9 as well as IL-1-receptor 1, and IFN-αßR on infection. Only mice lacking TLR7 exhibited a phenotype, with mortality intermediate between MyD88(-/-) and control mice with deficits in both the development of peripheral immunity and rabies virus clearance from the CNS.

Development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in humans.

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans.

Concepts in the pathogenesis of rabies.

Rabies is a zoonotic disease that remains an important public health problem worldwide and causes more than 70,000 human deaths each year. The causative agent of rabies is rabies virus (RV), a negative-stranded RNA virus of the rhabdovirus family. Neuroinvasiveness and neurotropism are the main features that define the pathogenesis of rabies. Although RV pathogenicity is a multigenic trait involving several elements of the RV genome, the RV glycoprotein plays a major role in RV pathogenesis by controlling the rate of virus uptake and trans-synaptic virus spread, and by regulating the rate of virus replication. Pathogenic street RV strains differ significantly from tissue culture-adapted RV strains in their neuroinvasiveness. Whereas street RV strains are highly neuroinvasive, most tissue culture-adapted RV strains have either no or only limited ability to invade the CNS from a peripheral site. The high neuroinvasiveness of pathogenic street RVs is, at least in part, due to their ability to evade immune responses and to conserve the structures of neurons. The finding that tissue culture-adapted RV strains replicate very fast and induce strong innate and adaptive immune responses opens new avenues for therapeutic intervention against rabies.