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

AbstractRabies 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

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

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

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.The National Institutes of Health grant ROIAI093666 (to M.F.), National Research Foundation, South Africa (UID 85786) and International Foundation for Science. J. Kgaladi’s PhD studies were supported by a freestanding bursary from the National Research Foundation, South Africa, the Poliomyelitis Research Foundation and the University of Pretoria study abroad bursary program.http://www.mdpi.com/journal/tropicalmedam2018Medical VirologyMicrobiology and Plant Patholog

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

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

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