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.

T-bet Is Required for the Rapid Clearance of Attenuated Rabies Virus from Central Nervous System Tissue.

Much of our understanding of CNS immunity has been gained from models involving pathological inflammation. Attenuated rabies viruses (RABV) are unique tools to study CNS immunity in the absence of conventional inflammatory mechanisms, as they spread from the site of inoculation to the CNS transaxonally, thereby bypassing the blood-brain barrier (BBB), and are cleared without neutrophil or monocyte infiltration. To better understand the role of CD4 T cell subsets in the clearance of the virus from CNS tissues, we examined the development of antiviral immunity in wild-type (WT) and T-bet knockout mice (T-bet(-/-)), which lack Th1 cells. Early control of RABV replication in the CNS tissues of WT mice is associated with the production of IFN-γ, with antiviral effects likely mediated through the enhanced expression of type I IFNs. Of interest, IFN-α and -γ are overexpressed in the infected T-bet(-/-) by comparison with WT CNS tissues, and the initial control of RABV infection is similar. Ultimately, attenuated RABV are cleared from the CNS tissues of WT mice by Ab locally produced by the activities of infiltrating T and B cells. Although T and B cell infiltration into the CNS of infected T-bet(-/-) mice is comparable, their activities are not, the consequence being delayed, low-level Ab production and prolonged RABV replication. More importantly, neither T-bet(-/-) mice immunized with an attenuated virus, nor WT mice with Th2 RABV-specific immunity induced by immunization with inactivated virus, are protected in the long term against challenge with a pathogenic RABV.

Differential Host Immune Responses after Infection with Wild-Type or Lab-Attenuated Rabies Viruses in Dogs.

METHODOLOGY/PRINCIPAL FINDINGS: The experimental infection of dogs with TriGAS induced high levels of VNA in the serum, whereas wt RABV infection did not. Dogs infected with TriGAS developed antibodies against the virus including its glycoprotein, whereas dogs infected with DRV-NG11 only developed rabies antibodies that are presumably specific for the nucleoprotein, (N) and not the glycoprotein (G). We show that infection with TriGAS induces early activation of B cells in the draining lymph nodes and persistent activation of DCs and B cells in the blood. On the other hand, infection with DRV-NG11 fails to induce the activation of DCs and B cells and further reduces CD4 T cell production. Further, we show that intrathecal (IT) immunization of TriGAS not only induced high levels of VNA in the serum but also in the CSF while intramuscular (IM) immunization of TriGAS induced VNA only in the serum. In addition, high levels of total protein and WBC were detected in the CSF of IT immunized dogs, indicating the transient enhancement of blood-brain barrier (BBB) permeability, which is relevant to the passage of immune effectors from periphery into the CNS. CONCLUSIONS/SIGNIFICANCE: IM infection of dogs with TriGAS induced the production of serum VNA whereas, IT immunization of TriGAS in dogs induces high levels of VNA in the periphery as well as in the CSF and transiently enhances BBB permeability. In contrast, infection with wt DRV-NG11 resulted in the production of RABV-reactive antibodies but VNA and antibodies specific for G were absent. As a consequence, all of the dogs infected with wt DRV-NG11 succumbed to rabies. Thus the failure to activate protective immunity is one of the important features of RABV pathogenesis in dogs.

Pre- and post-exposure safety and efficacy of attenuated rabies virus vaccines are enhanced by their expression of IFNγ.

Consistent with evidence of a strong correlation between interferon gamma (IFNγ) production and rabies virus (RABV) clearance from the CNS, we recently demonstrated that engineering a pathogenic RABV to express IFNγ highly attenuates the virus. Reasoning that IFNγ expression by RABV vaccines would enhance their safety and efficacy, we reverse-engineered two proven vaccine vectors, GAS and GASGAS, to express murine IFNγ. Mortality and morbidity were monitored during suckling mice infection, immunize/challenge experiments and mixed intracranial infections. We demonstrate that GASγ and GASγGAS are significantly attenuated in suckling mice compared to the GASGAS vaccine. GASγ better protects mice from lethal DRV4 RABV infection in both pre- and post-exposure experiments compared to GASGAS. Finally, GASγGAS reduces post-infection neurological sequelae, compared to control, during mixed intracranial infection with DRV4. These data show IFNγ expression by a vaccine vector can enhance its safety while increasing its efficacy as pre- and post-exposure treatment.

Expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon.

UNLABELLED: Previous animal model experiments have shown a correlation between interferon gamma (IFN-γ) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN-γ by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN-γ gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBNγ. Morbidity and mortality were monitored in mice infected intranasally with SPBNγ or SPBN(-) control virus to determine the degree of attenuation caused by the expression of IFN-γ. Incorporation of IFN-γ into the rabies virus genome highly attenuated the virus. SPBNγ has a 50% lethal dose (LD50) more than 100-fold greater than SPBN(-). In vitro and in vivo mouse experiments show that SPBNγ infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNAR(-/-)) cannot control the SPBNγ infection and rapidly die. These data suggest that IFN-γ production has antiviral effects in rabies, largely due to the induction of type I interferons. IMPORTANCE: Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN-γ production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN-γ triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN-γ directly in the infected tissue reduces rabies virus replication and spread, limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN-γ to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus.

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.

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.

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.

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.

Infection of monocytes or immature dendritic cells (DCs) with an attenuated rabies virus results in DC maturation and a strong activation of the NFkappaB signaling pathway.

To assess the potential role of dendritic cells (DCs) or monocytes in the development of a protective immune response, we infected human immature DCs or monocytes with a live rabies virus (RV) vaccine strain (SPBNGAS-GAS) and a pathogenic RV (DOG4). Both cell types were infected with SPBNGAS-GAS and DOG4 and both RVs were similarly potent in inducing maturation of immature DCs or monocytes. However, in contrast to DOG4, SPBNGAS-GAS induced very high levels of IFN-alpha1 mRNA in monocytes and DCs. Furthermore, at least 26 other genes related to the NFkappaB signaling pathway were strongly upregulated in SPBNGAS-GAS-infected DCs, but only somewhat increased in DOG4-infected cells. Thus, the extent of upregulation of NFkappaB pathway-related genes in DCs infected with the live RV vaccine strain might explain the strong protective activity of SPBNGAS-GAS.

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.

Dominance of a nonpathogenic glycoprotein gene over a pathogenic glycoprotein gene in rabies virus.

The nonpathogenic phenotype of the live rabies virus (RV) vaccine SPBNGAN is determined by an Arg-->Glu exchange at position 333 in the glycoprotein, designated GAN. We recently showed that after several passages of SPBNGAN in mice, an Asn-->Lys mutation arose at position 194 of GAN, resulting in GAK, which was associated with a reversion to the pathogenic phenotype. Because an RV vaccine candidate containing two GAN genes (SPBNGAN-GAN) exhibits increased immunogenicity in vivo compared to the single-GAN construct, we tested whether the presence of two GAN genes might also enhance the probability of reversion to pathogenicity. Comparison of SPBNGAN-GAN with RVs constructed to contain either both GAN and GAK genes (SPBNGAN-GAK and SPBNGAK-GAN) or two GAK genes (SPBNGAK-GAK) showed that while SPBNGAK-GAK was pathogenic, SPBNGAN-GAN and SPBNGAN-GAK were completely nonpathogenic and SPBNGAK-GAN showed strongly reduced pathogenicity. Analysis of genomic RV RNA in mouse brain tissue revealed significantly lower virus loads in SPBNGAN-GAK- and SPBNGAK-GAN-infected brains than those detected in SPBNGAK-GAK-infected brains, indicating the dominance of the nonpathogenic phenotype determined by GAN over the GAK-associated pathogenic phenotype. Virus production and viral RNA synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN-, and SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, suggesting control of GAN dominance at the level of viral RNA synthesis. These data point to the lower risk of reversion to pathogenicity of a recombinant RV carrying two identical GAN genes compared to that of an RV carrying only a single GAN gene.

A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice.

Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus.

Overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice.

The effect of tumor necrosis factor alpha (TNF-alpha) on rabies virus (RV) infection of the mouse central nervous system (CNS) was studied, using recombinant RV engineered to express either soluble TNF-alpha [SPBN-TNF-alpha+] or insoluble membrane-bound TNF-alpha [SPBN-TNF-alpha(MEM)]. Growth curves derived from infections of mouse neuroblastoma NA cells revealed significantly less spread and production of SPBN-TNF-alpha+ than of SPBN-TNF-alpha(MEM) or SPBN-TNF-alpha-, which carries an inactivated TNF-alpha gene. The expression of soluble or membrane-bound TNF-alpha was not associated with increased cell death or induction of alpha/beta interferons. Brains of mice infected intranasally with SPBN-TNF-alpha+ showed significantly less virus spread than did mouse brains after SPBN-TNF-alpha- infection, and none of the SPBN-TNF-alpha+-infected mice succumbed to RV infection, whereas 80% of SPBN-TNF-alpha- -infected mice died. Reduced virus spread in SPBN-TNF-alpha+-infected mouse brains was paralleled by enhanced CNS inflammation, including T-cell infiltration and microglial activation. These data suggest that TNF-alpha exerts its protective activity in the brain directly through an as yet unknown antiviral mechanism and indirectly through the induction of inflammatory processes in the CNS.

A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity.

Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu(333) remained unchanged after five mouse passages, an Asn(194)-->Lys(194) mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn(194) with Lys(194) in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn(194)-->Lys(194) mutation does not arise when Asn(194) is exchanged with Ser(194) (SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs.

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.

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.

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis.

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.

Identification of viral genomic elements responsible for rabies virus neuroinvasiveness.

Attenuated tissue culture-adapted and natural street rabies virus (RV) strains differ greatly in their neuroinvasiveness. To identify the elements responsible for the ability of an RV to enter the CNS from a peripheral site and to cause lethal neurological disease, we constructed a full-length cDNA clone of silver-haired bat-associated RV (SHBRV) strain 18 and exchanged the genes encoding RV proteins and genomic sequences of this highly neuroinvasive RV strain with those of a highly attenuated nonneuroinvasive RV vaccine strain (SN0). Analysis of the recombinant RV (SB0), which was recovered from SHBRV-18 cDNA, indicated that this RV is phenotypically indistinguishable from WT SHBRV-18. Characterization of the chimeric viruses revealed that in addition to the RV glycoprotein, which plays a predominant role in the ability of an RV to invade the CNS from a peripheral site, viral elements such as the trailer sequence, the RV polymerase, and the pseudogene contribute to RV neuroinvasiveness. Analyses also revealed that neuroinvasiveness of an RV correlates inversely with the time necessary for internalization of RV virions and with the capacity of the virus to grow in neuroblastoma cells.

In vitro growth and stability of recombinant rabies viruses designed for vaccination of wildlife.

Three live rabies virus (RV) recombinant vaccine candidates, SPBNGA, SPBNGA-Cyto c (+), and SPBNGA-GA, were examined for their production levels and stability. Maximum production levels up to 10(10) infectious particles/mL were achieved using bioreactor technology. All virus lots exhibited thermostability profiles typical for RV vaccines and were non-pathogenic for intracranially inoculated immunocompetent mice. Moreover, sequence analysis indicated high genetic stability in all three RVs during 10 consecutive passages in newborn mice. This analysis revealed no change in the extra RV G gene in the SPBNGA-GA vaccine or in the cytochrome c gene in the SPBNGA-Cyto c (+) vaccine. Moreover, no changes were detected in the G gene codon for Glu333, which renders the virus non-pathogenic. However, after the fifth passage, a mutation resulting in an Asn194 --> Lys194 exchange emerged in the G genes of all three RVs. This mutation was associated with a modest increase in pathogenicity in SPBNGA and SPBNGA-Cyto c (+), but not in SPBNGA-GA, which contained the mutation in only one of its two G genes and which remained non-pathogenic. These results demonstrate the feasibility of producing RV vaccines that remain highly stable even after multiple passages.