Recombinant Rabies Virus Research Articles

Dimerization of Rabies Virus Phosphoprotein and Phosphorylation of Its Nucleoprotein Enhance Their Binding Affinity

The dynamic interplay between a multimeric phosphoprotein (P) and polymeric nucleoprotein (N) in complex with the viral RNA is at the heart of the functioning of the RNA-synthesizing machine of negative-sense RNA viruses of the order Mononegavirales. P multimerization and N phosphorylation are often cited as key factors in regulating these interactions, but a detailed understanding of the molecular mechanisms is not yet available. Working with recombinant rabies virus (RABV) N and P proteins and using mainly surface plasmon resonance, we measured the binding interactions of full-length P dimers and of two monomeric fragments of either circular or linear N-RNA complexes, and we analyzed the equilibrium binding isotherms using different models. We found that RABV P binds with nanomolar affinity to both circular and linear N-RNA complexes and that the dimerization of P protein enhances the binding affinity by 15–30-fold as compared to the monomeric fragments, but less than expected for a bivalent ligand, in which the binding domains are connected by a flexible linker. We also showed that the phosphorylation of N at Ser389 creates high-affinity sites on the polymeric N-RNA complex that enhance the binding affinity of P by a factor of about 360.

A novel inactivated oral rabies vaccine with the incorporation of U-OMP19 enhances the immunogenicity by reducing viral proteins degradation and activating dendritic cells in a mouse model

Currently, dogs, especially stray dogs, and/or wild animals are the main sources of rabies transmission, and oral vaccination is the most practical way to control rabies in these animals. Safety and efficacy are two key criteria for developing oral vaccines. Concerning the efficacy of oral vaccines, degradation of immunogens by gastrointestinal fluid is a major challenge, resulting in suboptimal immune responses after vaccination. For safety reasons, inactivated vaccines are the most optimal choice. In the present study, a recombinant rabies virus (RABV) with un-lipidated outer membrane protein 19 (U-OMP19) of Brucella spp incorporated into RABV virions, designated as LBNSE-OMP19-G, was constructed and rescued. We found that U-OMP19 was incorporated into LBNSE-OMP19-G virion, which could protect RABV G protein from digestion by gastrointestinal fluids in vitro. Moreover, the immunogenicity of LBNSE-OMP19-G as an inactivated oral vaccine was evaluated, and the inactivated LBNSE-OMP19-G could activate more dendritic cells (DCs) and promote the generation of follicular helper T (TFH) cells, germinal center (GC) B cells, and plasma cells in immunized mice compared with those in mice immunized with parent virus LNBSE, which consequently induced a higher level of virus neutralizing antibody and provided better protection after a lethal challenge of rabies. These data indicate that LBNSE-OMP19-G, which has good safety and immunogenicity, could be a potential inactivated oral rabies vaccine candidate.

Analytical Methods for Evaluating the Immunogenicity of Recombinant Rabies Virus Glycoprotein Expressed in the Yeast Komagataella phaffii

ABSTRACT Background Rabies is a fatal viral disease preventable by vaccination. The multiple-dose regimens, along with the high production costs of current rabies vaccines, limit their use in rabies-endemic countries with developing economies and consequently there is a need for new efficacious, low-cost rabies vaccines. This study investigates the immunogenicity of recombinant rabies virus glycoprotein (rRABVG), expressed in the yeast Komagataella phaffii (K. phaffii), as a candidate subunit rabies vaccine. Methods Monoclonal antibodies were used to confirm neutralizing epitopes presence on the rRABVG. The rRABVG potency was estimated by antigen quantification methods using ELISA and SRID. Serological methods, specifically ELISA and RFFIT, were applied to investigate the immune response of mice groups immunized with rRABVG varying doses, with or without adjuvant. Results The potency estimated by antigen quantification was dependent on the method employed. Active immunization assessment using ELISA was effective when the solid-phase antigen is the rRABVG. The RFFIT data indicated that a single adjuvanted dose of 20 µg rRABVG is sufficient for virus-neutralizing antibodies induction at a protective level of 0.5 IU/mL within 10 days post immunization. Conclusion These data demonstrate that K. phaffii produced rRABVG is immunoactive and could be an attractive candidate to develop a low-cost subunit rabies vaccine.

Rabies virus-based oral and inactivated vaccines protect minks against SARS-CoV-2 infection and transmission1

Minks are highly susceptible to SARS-CoV-2, and have transmitted SARS-CoV-2 to humans. Oral immunization is one of the most promising strategies to prevent SARS-CoV-2 infection and transmission in minks. Here, we generated three recombinant rabies viruses (RABV), rERAG333E/S6P, rERAG333E/DS6P and rERAG333E/BA2S6P, expressing the prefusion-stabilized SARS-CoV-2 spike protein of wild-type (S6P), δ (DS6P) or BA.2 (BA2S6P) strain based on an oral rabies vaccine candidate (rERAG333E). Oral or inactivated immunization of the three RABVs monovalent or trivalent were safe, and induced robust RABV neutralizing antibody and cross-antibody responses against the three SARS-CoV-2 in mice and minks. The challenge tests showed that two doses of rERAG333E-S6P as an oral or inactivated vaccine completely protected mice against mouse-adapted SARS-CoV-2 infection in the upper and lower respiratory tracts, and largely prevented viral replication and lung damage caused by wild-type SARS-CoV-2 infection in minks. Notably, we also confirmed that two doses of rERAG333E-S6P as an oral or inactivated vaccine can largely protect minks against wild-type SARS-CoV-2 transmission via respiratory droplets. Our findings suggest that rERAG333E-based COVID-19 vaccines appear to be suitable oral candidates to protect minks from SARS-CoV-2 infection and transmission, and may serve as inactivated vaccines for further investigation in humans.

Construction of Vero cell-adapted rabies vaccine strain by five amino acid substitutions in HEP-Flury strain

Rabies virus (RABV) causes fatal neurological disease. Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) using inactivated-virus vaccines are the most effective measures to prevent rabies. In Japan, HEP-Flury, the viral strain, used as a human rabies vaccine, has historically been propagated in primary fibroblast cells derived from chicken embryos. In the present study, to reduce the cost and labor of vaccine production, we sought to adapt the original HEP-Flury (HEP) to Vero cells. HEP was repeatedly passaged in Vero cells to generate ten- (HEP-10V) and thirty-passaged (HEP-30V) strains. Both HEP-10V and HEP-30V grew significantly better than HEP in Vero cells, with virulence and antigenicity similar to HEP. Comparison of the complete genomes with HEP revealed three non-synonymous mutations in HEP-10V and four additional non-synonymous mutations in HEP-30V. Comparison among 18 recombinant HEP strains constructed by reverse genetics and vesicular stomatitis viruses pseudotyped with RABV glycoproteins indicated that the substitution P(L115H) in the phosphoprotein and G(S15R) in the glycoprotein improved viral propagation in HEP-10V, while in HEP-30V, G(V164E), G(L183P), and G(A286V) in the glycoprotein enhanced entry into Vero cells. The obtained recombinant RABV strain, rHEP-PG4 strain, with these five substitutions, is a strong candidate for production of human rabies vaccine.

Immunogenicity evaluation of a bivalent vaccine based on a recombinant rabies virus expressing gB protein of FHV-1 in mice and cats

Feline viral rhinotracheitis (FVR) is caused by the feline herpesvirus-1 (FHV-1), which commonly results in upper respiratory symptoms, and can result in death in the kittens and weak cats. Rabies is an infectious disease with zoonotic characteristics highly relevant to public health and also poses a serious threat to cats. Vaccines are the most effective method to control the spread of both FHV-1 and RABV and have the advantage that they produce long-term specific immune responses. In this study, we constructed a bivalent vaccine against FHV-1 and rabies virus (RABV) simultaneously. The vaccine was constructed by cloning FHV-1 gB into a RABV based vector, and the recombinant RABV (SRV9-FHV-gB) expressing the FHV-1 gB protein was rescued. The growth characteristics of SRV9-FHV-gB were analyzed on NA and BSR cells. To assess the immunogenicity of the vaccine, mice and cats were immunized with SRV9-FHV-gB supplemented with Gel02 adjuvant.The SRV9-FHV-gB exhibited the same growth characteristics as the parent virus SRV9 in both BSR cells and NA cells. The safety of SRV9-FHV-gB was evaluated using 5-day-old and 14-day-old suckling mice. The results showed that mice infected with the SRV9-FHV-gB survived for longer than those in the SRV9 group. Mice immunized with inactivated SRV9-FHV-gB produced high titers of specific antibodies against FHV-1 and neutralizing antibodies against RABV. Cats that received three immunizations with SRV9-FHV-gB also produced neutralizing antibodies against both FHV-1 and RABV. This study represents the first time that a bivalent vaccine targeting FHV-1 and RABV has been constructed, laying the foundations and providing inspiration for the development of other multivalent vaccines.

New rabies viral resources for multi-scale neural circuit mapping

Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer’s disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.

Lyssavirus M protein degrades neuronal microtubules by reprogramming mitochondrial metabolism.

Infection with neurotropic viruses may result in changes in host behavior, which are closely associated with degenerative changes in neurons. The lyssavirus genus comprises highly neurotropic viruses, including the rabies virus (RABV), which has been shown to induce degenerative changes in neurons, marked by the self-destruction of axons. The underlying mechanism by which the RABV degrades neuronal cytoskeletal proteins remains incomplete. In this study, we show that infection with RABV or overexpression of its M protein can disrupt mitochondrial metabolism by binding to Slc25a4. This leads to a reduction in NAD+ production and a subsequent influx of Ca2+ from the endoplasmic reticulum and mitochondria into the cytoplasm of neuronal cell lines, activating Ca2+-dependent proteinase calpains that degrade α-tubulin. We further screened the M proteins of different lyssaviruses and discovered that the M protein of the dog-derived RABV strain (DRV) does not degrade α-tubulin. Sequence analysis of the DRV M protein and that of the lab-attenuated RABV strain CVS revealed that the 57th amino acid is vital for M-induced microtubule degradation. We generated a recombinant RABV with a mutation at the 57th amino acid position in its M protein and showed that this mutation reduces α-tubulin degradation in vitro and axonal degeneration in vivo. This study elucidates the mechanism by which lyssavirus induces neuron degeneration.IMPORTANCEPrevious studies have suggested that RABV (rabies virus, the representative of lyssavirus) infection induces structural abnormalities in neurons. But there are few articles on the mechanism of lyssavirus' effect on neurons, and the mechanism of how RABV infection induces neurological dysfunction remains incomplete. The M protein of lyssavirus can downregulate cellular ATP levels by interacting with Slc25a4, and this decrease in ATP leads to a decrease in the level of NAD+ in the cytosol, which results in the release of Ca2+ from the intracellular calcium pool, the endoplasmic reticulum, and mitochondria. The presence of large amounts of Ca2+ in the cytoplasm activates Ca2+-dependent proteases and degrades microtubule proteins. The amino acid 57 of M protein is the key site determining its disruption of mitochondrial metabolism and subsequent neuron degeneration.

Inactivated Recombinant Rabies Virus Displaying the Nipah Virus Envelope Glycoproteins Induces Systemic Immune Responses in Mice.

Nipah virus (NiV) causes severe, lethal encephalitis in humans and pigs. However, there is no licensed vaccine available to prevent NiV infection. In this study, we used the reverse genetic system based on the attenuated rabies virus strain SRV9 to construct two recombinant viruses, rSRV9-NiV-F and rSRV9-NiV-G, which displayed the NiV envelope glycoproteins F and G, respectively. Following three immunizations in BALB/c mice, the inactivated rSRV9-NiV-F and rSRV9-NiV-G alone or in combination, mixed with the adjuvants ISA 201 VG and poly (I:C), were able to induce the antigen-specific cellular and Th1-biased humoral immune responses. The specific antibodies against rSRV9-NiV-F and rSRV9-NiV-G had reactivity with two constructed bacterial-like particles displaying the F and G antigens of NiV. These data demonstrate that rSRV9-NiV-F or rSRV9-NiV-G has the potential to be developed into a promising vaccine candidate against NiV infection.

Construction and Development of New Rabies Virus Vectors Carrying Enhanced Green Fluorescent Protein

Aim: Rabies is one of the most lethal viral diseases. This study developed a recombinant rabies virus, PVEGFP, expressing an EGFP protein based on PV strain to be applied in virus-neutralizing antibody (VNA) titration methods. Methods: The EGFP gene was inserted into the PVMCS strain, a previously discussed recombinant virus. The recombinant virus was applied in the rapid fluorescent focus inhibition test (RFFIT) VNA titration method, and six vaccinated persons were studied for validation. Results: The recombinant rabies strain showed similar propagation properties to its ancestry strain, PVMCS. It could also measure VNA titers in the RFFIT method not significantly different from those acquired using the CVS-11 virus. Conclusion: Utilizing the PVEGFP strain in the RFFIT method can produce rapid, reliable and affordable results.

Over-expression of IL-33 enhances rabies virus early antigen presentations and cellular immune responses in mice

Human inactivated rabies virus (RABV) vaccines have been widely used worldwide over 30 years. The mechanisms of humoral immunity elicited by previously reported rabies candidate vaccines have been fully investigated, but little is known about the cellular immunity profiles. Herein, the recombinant RABV rLBNSE-IL-33 overexpressing the mouse interleukin-33 (IL-33) proliferated well in Neuro-2a cells and had no effects with the parent virus on growth kinetic in vitro and viral pathogenicity in mice. The rLBNSE-IL-33 experienced more antigen presentations by MHC-II on DCs and activated more CD4+ T cells which helped recruit more CD19+CD40+ B cells in blood and promote rapid and robust IgG1 antibodies responses at initial infection stage compared with the parent rLBNSE strain. Simultaneously, the rLBNSE-IL-33 were also presented by MHC-I to CD8+ T cells which contributed to produce high levels of IgG2a. The rLBNSE-IL-33 elicited significantly high levels of RABV-specific IFN-γ secreting memory CD4+ T cells, more RABV-specific IL-4 and IFN-γ secreting memory CD8+ T cells in spleens at early infection stage in mice. Altogether, overexpression of IL-33 in rLBNSE-IL-33 enhanced early antigen presentation, markedly promote CD4+, memory CD4+ and CD8+ T cells-mediated responses and provided a 100 % protection from lethal RABV challenge in mice. These findings provided an alternative novel therapy and vaccine strategy in future.

Characterisation of a Live-Attenuated Rabies Virus Expressing a Secreted scFv for the Treatment of Rabies.

Rabies virus (RABV) causes possibly the oldest disease and is responsible for an estimated >59,000 human fatalities/year. Post exposure prophylaxis (PEP), the administration of vaccine and rabies immunoglobulin, is a highly effective tool which is frequently unavailable in RABV endemic areas. Furthermore, due to the constraints of the blood-brain barrier, current PEP regimes are ineffective after the onset of clinical symptoms which invariably result in death. To circumvent this barrier, a live-attenuated recombinant RABV expressing a highly RABV-neutralising scFv antibody (62-71-3) linked to the fluorescent marker mCherry was designed. Once rescued, the resulting construct (named RABV-62scFv) was grown to high titres, its growth and cellular dissemination kinetics characterised, and the functionality of the recombinant 62-71-3 scFv assessed. Encouraging scFv production and subsequent virus neutralisation results demonstrate the potential for development of a therapeutic live-attenuated virus-based post-infection treatment (PIT) for RABV infection.

A simplified method for measuring neutralising antibodies against rabies virus

Rabies virus (RABV) causes a fatal encephalitis that can be prevented through timely vaccination. The levels of virus neutralising antibodies against rabies virus induced by vaccination can be measured using the fluorescent antibody virus neutralisation (FAVN) test. Following incubation of live virus with sera, this method involves the fixation of cell monolayers and staining of rabies virus-specific antigen using fluorescein isothiocyanate (FITC) -conjugated antibody to enable visualisation of rabies virus antigen using a fluorescence microscope. To simplify this procedure, a fluorescent recombinant rabies virus was constructed using reverse genetics by inserting the gene for the mCherry fluorescent protein in front of the ribonucleoprotein gene of the SAD B-19 genome and replacing its glycoprotein with that of the Challenge Virus Standard (CVS)-11 RABV strain to ensure antigenic authenticity with the FAVN. This new recombinant virus (termed mCCCG) expressed the mCherry protein to high levels enabling direct observation of infected cells. In vitro growth kinetics of mCCCG were indistinguishable from that of CVS-11. The stability of the recombinant virus was assessed by sequencing several passages of the rescued virus and only minor changes were detected. Comparative assessment of the virus neutralisation test using mCherry producing virus (NTmCV) against the FAVN demonstrated that test results were equivalent to each other; therefore, mCCCG can be used as an alternative to CVS-11 for measuring antibody titres against the rabies virus. The use of NTmCV removes the need for expensive antibody conjugates and significantly reduces assay time. This would be particularly beneficial for RABV serological assessment in resource limited settings. Moreover, the reading of the plates can be automatically using a cell imaging reader.

A recombinant rabies virus chimera expressing the DC-targeting molecular MAB2560 shows enhanced vaccine immunogenicity through activation of dendritic cells.

Rabies, caused by the rabies virus (RABV), is an ancient and neglected zoonotic disease posing a large public health threat to humans and animals in developing countries. Immunization of animals with a rabies vaccine is the most effective way to control the epidemic and the occurrence of the disease in humans. Therefore, the development of cost-effective and efficient rabies vaccines is urgently needed. The activation of dendritic cells (DCs) is known to play an important role in improving the host immune response induced by rabies vaccines. In this study, we constructed a recombinant virus, rCVS11-MAB2560, based on the reverse genetic system of the RABV CVS11 strain. The MAB2560 protein (a DC-targeting molecular) was chimeric expressed on the surface of the viral particles to help target and activate the DCs when this virus was used as inactivated vaccine. Our results demonstrated that inactivated rCVS11-MAB2560 was able to promote the recruitment and/or proliferation of DC cells, T cells and B cells in mice, and induce good immune memory after two immunizations. Moreover, the inactivated recombinant virus rCVS11-MAB2560 could produce higher levels of virus-neutralizing antibodies (VNAs) in both mice and dogs more quickly than rCVS11 post immunization. In summary, the recombinant virus rCVS11-MAB2560 chimeric-expressing the molecular adjuvant MAB2560 can stimulate high levels of humoral and cellular immune responses in vivo and can be used as an effective inactivated rabies vaccine candidate.

Interleukin-1β suppresses rabies virus infection by activating cGAS-STING pathway and compromising the blood-brain barrier integrity in mice

Rabies, caused by rabies virus (RABV), is an ancient zoonotic disease that severely threatens the public health throughout the world. Previous study indicated that interleukin-1β (IL-1β) plays an important role in RABV infection. However, the mechanism how IL-1β affects RABV pathogenicity is still unknown yet. In this study, we confirmed that IL-1β was able to reduce viral titers of RABV in different cells, and the recombinant RABV expressing IL-1β, designated as rCVS-IL1β, could be suppressed in different cells due to the expression of IL-1β. Furthermore, the survival rates of mice infected with rCVS-IL1β by intramuscular route was significantly higher than those of mice infected with parent virus rCVS, which is associated with the less viral loads for entry into the central nervous system (CNS). We further characterized that the cGAS-STING pathway was activated in rCVS-IL1β infected bone marrow derived dendritic cells (BMDC), which could contribute to the decreased viral loads of RABV after intramuscular infection. Moreover, we also observed that the expression of IL-1β by rCVS-IL1β could compromise the blood-brain barrier (BBB) integrity by degrading the tight junction proteins, which allowing peripheral inflammatory cytokines, chemokines, and CD4+T cells to enter into the brain for the clearance of RABV in the CNS. Together, our study suggests that IL-1β could attenuate RABV pathogenicity through activating cGAS-STING pathway in to decrease the viral entry into the CNS and enhance the BBB permeability to promote RABV clearance in the CNS as well, which provides new insight into developing effective therapeutics for rabies.

Effects of adjuvants in a rabies-vectored Ebola virus vaccine on protection from surrogate challenge

Ebola virus is the primary contributor to the global threat of filovirus severe hemorrhagic fever, and Ebola virus disease has a case fatality rate of 50–90%. An inactivated, bivalent filovirus/rabies virus vaccine, FILORAB1, consists of recombinant rabies virus virions expressing the Ebola virus glycoprotein. FILORAB1 is immunogenic and protective from Ebola virus challenge in mice and non-human primates, and protection is enhanced when formulated with toll-like receptor 4 agonist Glucopyranosyl lipid adjuvant (GLA) in a squalene oil-in-water emulsion (SE). Through an adjuvant comparison in mice, we demonstrate that GLA-SE improves FILORAB1 efficacy by activating the innate immune system and shaping a Th1-biased adaptive immune response. GLA-SE adjuvanted mice and those adjuvanted with the SE component are better protected from surrogate challenge, while Th2 alum adjuvanted mice are not. Additionally, the immune response to FILORAB1 is long-lasting, as exhibited by highly-maintained serum antibody titers and long-lived cells in the spleen and bone marrow.

An inactivated recombinant rabies virus chimerically expressed RBD induces humoral and cellular immunity against SARS-CoV-2 and RABV

Many studies suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect various animals and transmit among animals, and even to humans, posing a threat to humans and animals. There is an urgent need to develop inexpensive and efficient animal vaccines to prevent and control coronavirus disease 2019 (COVID-19) in animals. Rabies virus (RABV) is another important zoonotic pathogen that infects almost all warm-blooded animals and poses a great public health threat. The present study constructed two recombinant chimeric viruses expressing the S1 and RBD proteins of the SARS-CoV-2 Wuhan01 strain based on a reverse genetic system of the RABV SRV9 strain and evaluated their immunogenicity in mice, cats and dogs. The results showed that both inactivated recombinant viruses induced durable neutralizing antibodies against SARS-CoV-2 and RABV and a strong cellular immune response in mice. Notably, inactivated SRV-nCoV-RBD induced earlier antibody production than SRV-nCoV-S1, which was maintained at high levels for longer periods. Inactivated SRV-nCoV-RBD induced neutralizing antibodies against both SARS-CoV-2 and RABV in cats and dogs, with a relatively broad-spectrum cross-neutralization capability against the SARS-CoV-2 pseudoviruses including Alpha, Beta, Gamma, Delta, and Omicron, showing potential to be used as a safe bivalent vaccine candidate against COVID-19 and rabies in animals.

A rabies virus-vectored vaccine expressing two copies of the Marburg virus glycoprotein gene induced neutralizing antibodies against Marburg virus in humanized mice

ABSTRACT Marburg virus disease (MVD) is a lethal viral haemorrhagic fever caused by Marburg virus (MARV) with a case fatality rate as high as 88%. There is currently no vaccine or antiviral therapy approved for MVD. Due to high variation among MARV isolates, vaccines developed against one strain fail to protect against other strains. Here we report that three recombinant rabies virus (RABV) vector vaccines encoding two copies of GPs covering both MARV lineages induced pseudovirus neutralizing antibodies in BALB/c mice. Furthermore, high-affinity human neutralizing antibodies were isolated from a humanized mouse model. The three vaccines produced a Th1-biased serological response similar to that of human patients. Adequate sequential immunization enhanced the production of neutralizing antibodies. Virtual docking suggested that neutralizing antibodies induced by the Angola strain seemed to be able to hydrogen bond to the receptor-binding site (RBS) in the GP of the Ravn strain through hypervariable regions 2 (CDR2) and CDR3 of the VH region. These findings demonstrate that three inactivated vaccines are promising candidates against different strains of MARV, and a novel fully humanized neutralizing antibody against MARV was isolated.

Construction of Recombinant Rabies Virus Vectors Expressing H or F Protein of Peste des Petits Ruminants Virus.

Simple SummaryPeste des petits ruminants (PPR) is one of the most contagious and fatal diseases of small ruminants. In this study, two recombinant viruses rSRV9-H and rSRV9-F, which express the envelope glycoprotein H (hemagglutinin protein) or F (fusion protein) protein, respectively, were successfully generated with a rabies virus as vector. The constructed viruses had good proliferative activity and stability and provided potential bivalent inactivated vaccine candidate strains for the prevention of PPR and livestock rabies.Peste des petits ruminants (PPR) is one of the most contagious and fatal diseases of small ruminants in the world and is classified as a category A epidemic disease. It is the target of a global eradication campaign led by the Office International des Epizooties (OIE) and Food and Agriculture Organization of the United Nations (FAO). The PPR live attenuated vaccine is currently the most widely used and approved vaccine, but the use of this vaccine interferes with the serological testing of the PPR elimination program, and there is a potential safety risk. Viral vector vaccines are one of the most promising methods to solve this dilemma. In this study, the full-length infectious clone plasmid of rabies virus (RABV), pD-SRV9-PM-LASV, was used as the backbone, and the envelope glycoprotein H (hemagglutinin protein) or F (fusion protein) gene of PPRV was inserted into the backbone plasmid to construct the infectious clones pD-SRV9-PM-PPRV-H and pD-SRV9-PM-PPRV-F, which express the PPRV H and PPRV F genes, respectively. The correct construction of these infectious clones was verified after sequencing and double digestion. The infectious clones were transfected with a helper plasmid into BSR/T7 cells, and recombinant viruses were successfully rescued by direct immunofluorescence, indirect immunofluorescence, Western blotting, and transmission electron microscopy and named rSRV9-H and rSRV9-F. The results of growth kinetics studies indicated that the inserted gene did not affect virus proliferation. Stability studies revealed that the inserted target gene was stably expressed in recombinant RABV for at least 15 generations. In this study, the recombinant viruses rSRV9-H and rSRV9-F were successfully rescued. The constructed viruses had good proliferative activity and stability and provided potential bivalent inactivated vaccine candidate strains for the prevention of PPR and livestock rabies.

Interleukin-25 enhances humoral immune responses caused by the rabies virus

ABSTRACT Rabies is an important zoonotic disease caused by the rabies virus (RABV). Currently, no effective treatment is available for this condition. The prevention and control of rabies mainly depend on effective vaccination. Therefore, it is crucial to enhance the immune responses induced by the rabies vaccine. Virus neutralizing antibodies (VNA) induced by rabies vaccines are important for the clearance of RABV. Interleukin-25 (IL-25) has been demonstrated to activate T helper type 2 cells that contribute to humoral immune responses. The IL-25 gene was inserted into the genome of RABV, and the immunogenicity of recombinant RABV with IL-25 gene was investigated to develop more efficient rabies vaccines. Here, we found that the expression of IL-25 did not affect RABV production in vitro and pathogenicity in vivo. However, recombinant RABV expression of IL-25 induced a better VNA level than the parental virus in mice. In addition, expression of IL-25 enhanced the IgG1 level induced by RABV. Furthermore, mice immunized with recombinant RABV showed a higher survival rate and milder clinical signs than those immunized with the parent strain after challenge with CVS-11. Thus, these results showed that IL-25 could enhance the humoral immune responses induced by RABV, suggesting that IL-25 can be used as a viral vaccine adjuvant.