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1.  A Novel Rabies Vaccine Based on a Recombinant Parainfluenza Virus 5 Expressing Rabies Virus Glycoprotein 
Journal of Virology  2013;87(6):2986-2993.
Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD50) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 106 PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 108 PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 108 PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.
doi:10.1128/JVI.02886-12
PMCID: PMC3592160  PMID: 23269806
2.  Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection. 
Journal of Virology  1991;65(6):3400-3405.
Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.
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PMCID: PMC241005  PMID: 2033678
3.  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 
Journal of Virology  2013;87(3):1834-1841.
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.
doi:10.1128/JVI.02589-12
PMCID: PMC3554143  PMID: 23192867
4.  A single immunization with recombinant rabies virus (ERAG3G) confers complete protection against rabies in mice 
Purpose
New alternative bait rabies vaccines applicable to pet dogs and wild animals are needed to eradicate rabies in Korea. In this study, recombinant rabies virus, ERAG3G strain was constructed using reverse genetic system and the safety, efficacy and immunogenicity of the ERAG3G strain was evaluated in mice and dogs.
Materials and Methods
Using the full-length genome mutated amino acid at position 333 of glycoprotein of rabies virus (RABV) and helper plasmids, the ERAG3G strain was rescued in BHK/T7-9 cells successfully. Mice were inoculated with the ERAG3G strain for safety and efficacy. Safety and immunogenicity of the dog inoculated with the ERAG3G strain (1 mL, 108.0 FAID50/mL) via intramuscular route was evaluated for 28 days after inoculation.
Results
The ERAG3G strain rescued by reverse genetic system was propagated well in the mouse neuroblastoma cells revealing titer of 108.5 FAID50/mL and was not pathogenic to 4- or 6-week-old mice that received by intramuscular or intracranical route. Immunization with the ERAG3G strain conferred complete protection from lethal RABV in mice. Dogs inoculated with the vaccine candidate via intramuscular route showed high neutralizing antibody titer ranging from 2.62 to 23.9 IU/mL at 28 days postinoculation.
Conclusion
Our findings suggest that the ERAG3G strain plays an important role in inducing protective efficacy in mice and causes to arise anti-rabies neutralizing antibody in dogs.
doi:10.7774/cevr.2014.3.2.176
PMCID: PMC4083070  PMID: 25003091
Rabies virus; Recombinant rabies virus; Vaccine; Animals
5.  A New Rabies Vaccine Based on a Recombinant Orf Virus (Parapoxvirus) Expressing the Rabies Virus Glycoprotein 
Journal of Virology  2013;87(3):1618-1630.
The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (107 PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals.
doi:10.1128/JVI.02470-12
PMCID: PMC3554190  PMID: 23175365
6.  Studies of ERA/BHK-21 rabies vaccine in skunks and mice. 
ERA rabies vaccine virus grown in BHK-21 13S cells (ERA/BHK-21) and street rabies virus were titrated in mice by intracerebral, intranasal and intramuscular inoculation. Mice were also given undiluted ERA/BHK-21 in baits. Skunks were given undiluted ERA/BHK-21 in baits and by intramuscular, intranasal and intestinal inoculation. Virus neutralizing antibody titers against rabies virus were measured over a three month observation period. The surviving skunks were challenged by intramuscular inoculation with rabies street virus from a skunk salivary gland suspension. When titrated in mice, ERA/BHK-21 had titers of 10(7.0), 10(5.2) and 10(3.9) median lethal doses per mL by the intracerebral, intranasal and intramuscular routes, respectively. All skunks (8/8) inoculated intranasally developed paralytic rabies by 12 days after exposure to ERA/BHK-21 virus. None of the skunks that developed vaccine-induced rabies had infectious virus in the submandibular salivary glands. Vaccine-induced rabies also occurred in 1/8 skunks in the intramuscularly inoculated group and in 1/8 in the intestinally inoculated group. The survival rates of challenged skunks in the various groups were as follows: intramuscular, 7/7; intestinal, 2/7; bait, 0/8; and control, 0/8. These results indicate that ERA/BHK-21 virus has a significant residual pathogenicity in mice and in skunks by some routes of inoculation. Skunks given vaccine intramuscularly were protected against challenge, while those skunks given the vaccine in baits were not.
PMCID: PMC1255401  PMID: 3280109
7.  Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro. 
Journal of Virology  1993;67(10):6080-6086.
We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not challenged with street rabies virus had anti-rabies virus activity. In vivo studies illustrated that mice inoculated in the footpad with preincubated mixtures of anti-N sera and virus were protected. In addition, anti-N sera inoculated into the site of virus challenge protected mice. The antiviral activity of anti-N sera was also demonstrated in vitro. Infectious virus was not detected in cultures 24 h following infection with virus that had been preincubated with anti-N sera. At later time points, infectious virus was detected, but inhibition of viral production was consistently > or = 99% compared with control cultures. The protective and antiviral inhibitory activity of the anti-N sera was identified as anti-N antibody by several methods. First, absorption of anti-N sera with goat anti-mouse immunoglobulin serum, but not normal goat serum, removed the activity. Second, radioimmuno-precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sucrose density gradient-fractionated anti-N sera showed that antiviral activity was present only in the fraction containing anti-N antibody. Finally, absorption of anti-N sera with insect cells infected with a baculovirus expressing the N protein removed the protective activity. These data indicate that anti-N antibody is a component of the resistance to rabies virus infections.
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PMCID: PMC238029  PMID: 8371354
8.  Immune Modulating Effect by a Phosphoprotein-deleted Rabies Virus Vaccine Vector Expressing Two Copies of the Rabies Virus Glycoprotein Gene 
Vaccine  2008;26(50):6405-6414.
The type of immune response induced by a vaccine is a critical factor that determines its effectiveness in preventing infection or disease. Inactivated and live rabies virus (RV) vaccine strains elicit an IgG1-biased and IgG1/IgG2a-balanced antibody response, respectively. However, IgG2a antibodies are potent inducers of anti-viral effector functions, and therefore, a viral vaccine vector that can elicit an IgG2a-biased antibody response may be more effective against RV infection. Here we describe the humoral immune response of a live replication-deficient phosphoprotein (P)-deleted RV vector (SPBN-ΔP), or a recombinant P-deleted virus that expresses two copies of the RV glycoprotein (G) gene (SPBN-ΔP-RVG), and compare it to a UV-inactivated RV. Mice inoculated with UV-inactivated RV induced predominantly an IgG1-specific antibody response, while live recombinant SPBN-ΔP exhibited a mixed IgG1/IgG2a antibody response, which is consistent with the isotype profiles from the replication-competent parental viruses. Survivorship in mice after pathogenic RV challenge indicates a ten-fold higher efficiency of live SPBN-ΔP compared to UV-inactivated SPBN-ΔP. In addition, SPBN-ΔP-RVG induced a more rapid and robust IgG2a response that protected mice more effectively than SPBN-ΔP. Of note, 103 ffu of SPBN-ΔP-RVG induced anti-RV antibodies that were 100% protective in mice against pathogenic RV challenge. The increased immune response was directed not only against RV G but also against the ribonucleoprotein (RNP), indicating that the expression of two RV G genes from SPBN-ΔP-RVG enhances the immune response to other RV antigens as well. In addition, Rag2 mice inoculated intramuscularly with 105 ffu/mouse of SPBN-ΔP showed no clinical signs of rabies, and no viral RNA was detected in the spinal cord or brain of inoculated mice. Therefore, the safety of the P-deleted vectors along with the onset and magnitude of the IgG2a-induced immune response by SPBN-ΔP-RVG indicate that this vector holds great promise as either a therapeutic or preventative vaccine against RV or other infectious diseases.
doi:10.1016/j.vaccine.2008.08.069
PMCID: PMC2629409  PMID: 18804506
rabies virus; replication-deficient; viral vector; isotypes; antibody subclass; vaccine; phosphoprotein; post-exposure prophylaxis
9.  Use of mouse anti-rabies monoclonal antibodies in postexposure treatment of rabies. 
Journal of Clinical Investigation  1989;84(3):971-975.
Immunization of mice and hamsters with a cocktail of mouse MAbs specific for rabies virus nucleocapsid protein and glycoprotein protected animals not only when challenged with a lethal dose of rabies virus after immunization, but also in post-exposure situations. Hamsters treated with the MAb cocktail 3 h after virus inoculation were completely protected from lethal rabies virus infection, and 80% of the animals survived when the MAb cocktail was given 36 h after virus challenge. The potential usefulness of this MAb cocktail for the postexposure treatment of human rabies is discussed.
PMCID: PMC329743  PMID: 2760222
10.  Zonal-Centrifuged Purified Duck Embryo Cell Culture Rabies Vaccine for Human Vaccination 
Applied Microbiology  1971;22(3):358-365.
Rabies virus produced in duck embryo cell culture was concentrated from volumes of 14 to 30 liters to 400 to 800 ml by zonal centrifugation. Virus titers of peak fractions were from 100- to 1,000-fold greater than those of the starting material. Vaccines were prepared by combining fractions with peak virus titers and diluting back to 10 times concentration. The resulting β-propiolactone-inactivated vaccines, when prepared as lyophilized vaccines with AlPO4 adjuvant diluents, were low in protein nitrogen (0.01 mg/ml), and three of four lots passed the National Institutes of Health potency test when tested as equivalent to a standard 10% suspension of duck embryo or mouse brain tissue vaccine. These vaccines also induced good sero-conversion in adult rabbits after a single 1-ml dose of vaccine. Guinea pigs sensitized with zonal-centrifuged purified duck embryo vaccine (with AlPO4 adjuvant) did not exhibit anaphylactic shock reactions when challenged with homologous vaccine. Also, no anaphylactic shock reactions were observed when guinea pigs were sensitized with either a 10% experimental duck embryo vaccine or cell culture vaccine and then challenged with the zonal-purified vaccine. However, guinea pigs sensitized with cell culture or zonal-purified vaccine and then challenged with the 10% experimental vaccine did show slight transitory congestion. The 10% experimental whole duck embryo vaccine was responsible for all observed anaphylactic shock reactions whether homologous or heterologous.
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PMCID: PMC376314  PMID: 5119204
11.  Inactivated or Live-Attenuated Bivalent Vaccines That Confer Protection against Rabies and Ebola Viruses ▿  
Journal of Virology  2011;85(20):10605-10616.
The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.
doi:10.1128/JVI.00558-11
PMCID: PMC3187516  PMID: 21849459
12.  The adaptation of a CTN-1 rabies virus strain to high-titered growth in chick embryo cells for vaccine development 
Virology Journal  2014;11:85.
Background
Rabies virus is the causative agent of rabies, a central nervous system disease that is almost invariably fatal. Currently vaccination is the most effective strategy for preventing rabies, and vaccines are most commonly produced from cultured cells. Although the vaccine strains employed in China include CTN, aG, PM and PV, there are no reports of strains that are adapted to primary chick embryo cells for use in human rabies prevention in China.
Results
Rabies virus strain CTN-1 V was adapted to chick embryo cells by serial passage to obtain the CTNCEC25 strain. A virus growth curve demonstrated that the CTNCEC25 strain achieved high titers in chick embryo cells and was nonpathogenic to adult mice by intracerebral inoculation. A comparison of the structural protein genes of the CTNCEC25 strain and the CTN-1 V strain identified eight amino acid changes in the mature M, G and L proteins. The immunogenicity of the CTNCEC25 strain increased with the adaptation process in chick embryo cells and conferred high protective efficacy. The inactivated vaccine induced high antibody responses and provided full protection from an intramuscular challenge in adult mice.
Conclusions
This is the first description of a CTNCEC25 strain that was highly adapted to chick embryo cells, and both its in vitro and in vivo biological properties were characterized. Given the high immunogenicity and good propagation characteristics of the CTNCEC25 strain, it has excellent potential to be a candidate for development into a human rabies vaccine with high safety and quality characteristics for controlling rabies in China.
doi:10.1186/1743-422X-11-85
PMCID: PMC4023167  PMID: 24885666
Rabies virus; CTN-1 V; Chick embryo cells; Adaptation; Vaccine
13.  Enhancement of immunogenicity and efficacy of a plasmid DNA rabies vaccine by nanoformulation with a fourth-generation amine-terminated poly(ether imine) dendrimer 
Purpose
Delayed onset of, and low magnitude of, protective immune responses are major drawbacks limiting the practical utility of plasmid vaccination against rabies. In this study we evaluated whether nanoformulation with the novel poly(ether imine) (PETIM) dendrimer can enhance the immunogenicity and efficacy of a plasmid-based rabies vaccine.
Materials and methods
A plasmid vaccine construct (pIRES-Rgp) was prepared by cloning the full-length rabies virus glycoprotein gene into pIRES vector. Drawing upon the results of our previous study, a dendriplex (dendrimer-DNA complex) of pIRES-Rgp was made with PETIM dendrimer (10:1 w/w, PETIM:pIRES-Rgp). In vitro transfection was done on baby hamster kidney (BHK)-21 cells to evaluate expression of glycoprotein gene from pIRES-Rgp and PETIM-pIRES-Rgp. Subsequently, groups of Swiss albino mice were immunized intramuscularly with pIRES-Rgp or PETIM-pIRES-Rgp. A commercially available cell culture rabies vaccine was included for comparison. Rabies virus neutralizing antibody (RVNA) titers in the immune sera were evaluated on days 14, 28, and 90 by rapid fluorescent focus inhibition test. Finally, an intracerebral challenge study using a challenge virus standard strain of rabies virus was done to evaluate the protective efficacy of the formulations.
Results
Protective levels of RVNA titer (≥0.5 IU/mL) were observed by day 14 in animals immunized with pIRES-Rgp and its dendriplex. Notably, PETIM-pIRES-Rgp produced 4.5-fold higher RVNA titers compared to pIRES-Rgp at this time point. All mice immunized with the PETIM-pIRES-Rgp survived the intracerebral rabies virus challenge, compared with 60% in the group which received pIRES-Rgp.
Conclusion
Our results suggest that nanoformulation with PETIM dendrimer can produce an earlier onset of a high-titered protective antibody response to a plasmid-based rabies vaccine. PETIM dendriplexing appears to be an efficacious nonviral delivery strategy to enhance genetic vaccination.
doi:10.2147/IJN.S53415
PMCID: PMC3912024  PMID: 24501540
nonviral gene delivery; rabies prophylaxis
14.  Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿  
Journal of Virology  2010;85(4):1634-1644.
Our previous studies indicated that recruitment and/or activation of dendritic cells (DCs) is important in enhancing the protective immune responses against rabies virus (RABV) (L. Zhao, H. Toriumi, H. Wang, Y. Kuang, X. Guo, K. Morimoto, and Z. F. Fu, J. Virol. 84:9642-9648). To address the importance of DC activation for RABV vaccine efficacy, the genes for several DC recruitment and/or activation molecules, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein 1α (MIP-1α), were individually cloned into RABV. The ability of these recombinant viruses to activate DCs was determined in vitro and in vivo. Infection of mouse bone marrow-derived DCs with each of the recombinant viruses resulted in DC activation, as shown by increased surface expression of CD11c and CD86 as well as an increased level of alpha interferon (IFN-α) production compared to levels observed after infection with the parent virus. Intramuscular infection of mice with each of the viruses recruited and/or activated more DCs and B cells in the periphery than infection with the parent virus, leading to the production of higher levels of virus-neutralizing antibodies. Furthermore, a single immunization with recombinant RABV expressing GM-CSF or MDC protected significantly more mice against intracerebral challenge with virulent RABV than did immunization with the parental virus. Yet, these viruses did not show more virulence than the parent virus, since direct intracerebral inoculation with each virus at up to 1 × 107 fluorescent focus units each did not induce any overt clinic symptom, such as abnormal behavior, or any neurological signs. Together, these data indicate that recombinant RABVs expressing these molecules activate/recruit DCs and enhance protective immune responses.
doi:10.1128/JVI.01552-10
PMCID: PMC3028913  PMID: 21106736
15.  Genetic control of serum neutralizing-antibody response to rabies vaccination and survival after a rabies challenge infection in mice. 
Journal of Virology  1986;59(1):98-102.
Quantitative differences in serum neutralizing-antibody (SNAb) responses to rabies vaccination and survival after a rabies challenge infection between two inbred mice strains, C3H/J and C57BL/6J, were shown to be under genetic control. A 99% confidence limit calculated from the SNAb response titers of 14 C57BL/6J mice resulted in an upper limit for the SNAb response titer of C57BL/6J mice at 50.63. A SNAb titer less than or equal to 50.63 in response to rabies vaccination was assigned the phenotype of hyporesponder, and a SNAb titer greater than 50.63 in response to rabies vaccination was assigned the phenotype of hyperresponder in this study. The hyper-SNAb response to rabies vaccination and the higher frequency of survival after rabies challenge infection behave as Mendelian dominant alleles in F1 hybrids (C3H/J X C57BL/6J) and backcross (BC) (F1 [C3H/J X C57BL/6J] X C57BL/6J) progeny. Both a relatively hyper-SNAb response and a higher frequency of vaccine-inducible survival phenotypes occur in C3H/J mice. On the other hand, both the relatively hypo-SNAb response and a lower frequency of vaccine-inducible survival phenotypes behave as Mendelian recessive alleles and occur in C57BL/6J mice. C3H/J mice are H-2 Kk, and C57BL/6J mice are H-2 Kb. All three phenotypic traits (H-2 type, SNAb response, and survival after rabies challenge infection) segregate as independent (unlinked) monogenic traits in BC progeny (F1 [C3H/J X C57BL/6J] X C57BL/6J). The genetically controlled survival trait is inducible by rabies vaccination, but SNAb response is not a parameter that measures successful vaccine induction of preexposure protection from a rabies challenge infection in the BC progeny. The essential role of vaccination in developing preexposure protection in genetically responsive mice is confirmed, but indicates that in vitro measurements other than SNAb titers need to be developed to identify mice that have failed to achieve preexposure protection by rabies vaccination. This study confirms Lodmell's findings (D. L. Lodmell and B. Chesebro, J. Virol. 50:359-362, 1984; D. L. Lodmell, J. Exp. Med. 157:451-460, 1983) that susceptibility to rabies infection is genetically controlled in some mice strains. Additionally, this study indicates that conventional rabies vaccination even with more potent vaccines may not induce protection from infection in some genetically susceptible individuals.
PMCID: PMC253043  PMID: 3086569
16.  Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein 
Vaccine  2012;30(43):6136-6141.
We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing ebolavirus (EBOV) glycoprotein (GP) that induce humoral immunity against each virus and confer protection from both lethal RABV and mouse-adapted EBOV challenge in mice. Here, we expand our investigation of the immunogenic properties of these bivalent vaccines in mice. Both live and killed vaccines induced primary EBOV GP-specific T-cells and a robust recall response as measured by interferon-γ ELISPOT assay. In addition to cellular immunity, an effective filovirus vaccine will likely require a multivalent humoral immune response against multiple virus species. As a proof-of-principle experiment, we demonstrated that inactivated RV-GP could be formulated with another inactivated RABV vaccine expressing the nontoxic fragment of botulinum neurotoxin A heavy chain (HC50) without a reduction in immunity to each component. Finally, we demonstrated that humoral immunity to GP could be induced by immunization of mice with inactivated RV-GP in the presence of pre-existing immunity to RABV. The ability of these novel vaccines to induce strong humoral and cellular immunity indicates that they should be further evaluated in additional animal models of infection.
doi:10.1016/j.vaccine.2012.07.073
PMCID: PMC3434297  PMID: 22884661
Ebola virus; rabies virus; vaccine; T cell; multivalent; platform
17.  Investigating the Role for IL-21 in Rabies Virus Vaccine-induced Immunity 
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R−/−) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R−/− mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R−/− mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R−/− mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.
Author Summary
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving post-exposure treatment. A person, disproportionately a child, dies of rabies every 20 minutes and the cost of rabies prevention exceeds $1 billion US dollars per year. The development of a single-dose human rabies vaccine would greatly reduce the burden of rabies globally by lowering the cost associated with rabies vaccination and saving lives. Understanding how B cells develop to produce protective virus neutralizing antibodies would greatly help to achieve the goal of developing a single-dose vaccine. In this report, we show that IL-21 is critical for the induction of primary vaccine-induced anti-RABV G antibody titers and that the effects of IL-21 are highly dependent on the dose of vaccine administered. In our model of rabies immunogenicity and protection, the lack of IL-21 receptor influenced the detection of B cells in germinal centers in lymph nodes or of plasma cells in bone marrow after immunization with low or high doses of vaccine, respectively. Overall, these preliminary results indicate that IL-21 has the potential to influence B cell development and functions in the context of rabies vaccine-induced immunity and protection.
doi:10.1371/journal.pntd.0002129
PMCID: PMC3597479  PMID: 23516660
18.  Immune Response in Primates Vaccinated with Duck Embryo Cell Culture Rabies Vaccine 
Applied Microbiology  1973;25(3):327-331.
Adult rhesus monkeys (Macaca mulata) were vaccinated with four inactivated rabies vaccines, including two cell culture vaccines, one zonal purified cell culture vaccine, and a 10% extracted duck embryo vaccine. The vaccines were potency tested by both National Institutes of Health (NIH) and Habel methods and passed one or both tests. However, a vaccine having acceptable potency by one method frequently failed or was marginal by the other procedure. Groups of three monkeys were inoculated with each vaccine by one of two schedules. The first consisted of four weekly 1-ml doses followed by a 1-ml booster dose at 6 months, and the second consisted of seven daily 1-ml doses of vaccine with no booster. Both zonal purified and extracted duck embryo vaccines induced detectable neutralizing antibody by day 7 with either schedule, and antibody titers elicited by the cell culture vaccine remained high through 210 days. However, antibody titers produced by the 10% duck embryo vaccine dropped sharply after their 28-day peak. Duck embryo cell culture vaccines with low or marginal potency as measured by Habel or NIH tests still produced rapid, high levels of serum-neutralizing antibody in primates. LD50 or NIH and Habel tests as measured in mice were not necessarily good indices of antibody response in the primate host. The need for a cell culture potency test that will yield a more predictable correlation with the definitive host's antibody response is discussed.
PMCID: PMC380805  PMID: 4633422
19.  The Activity of Rabies Vaccines against Genetic Clusters of Rabies Virus Circulating at the Territory of Ukraine 
Objective
To identify the presence of genetic clusters of rabies virus at the territory of Ukraine and to determine the degree of activity of rabies vaccines against these genetic clusters.
Introduction
To develop and implement an effective program of rabies eradication in Ukraine in 2008 was founded the unique collection of samples of pathological materials confirmed as positive in rabies at the regional veterinary laboratories of Ukraine. The collection is constantly updated and to present moment it includes 1389 samples from all regions of Ukraine, selected from 17 animal species and humans.
Methods
Identification of the rabies virus in samples of pathological material for their further selection was carried out using the test developed by us which based on RT-PCR with primers complementary to the conservative fragments of the 5’-end of nucleoprotein gene of rabies virus.
For the study of the street rabies virus isolates from the collection we use RT-PCR with the primers pair (509, 304) flanking the variable 3’-end part of nucleoprotein gene of the reference strain of rabies virus CVS (fragment in 377 bp).
Studies of rabies vaccines activity were carried out with modified method of U.S. National Institutes of Health using rabies virus street isolates of both genetic clusters instead of the Challenge Virus Standard (CVS). All isolates of street rabies virus were inoculated in a dose of 5–50 LD50. The criteria for evaluation of protective activity of rabies vaccine was effective dose (− lg ED50).
Results
In molecular genetic studies with variant-specific primers we established the presence in Ukraine of two clusters of rabies virus. Clusters I circulates on the right bank of the Dnipro river (the largest water barrier that divides the country into eastern and western side), and cluster II – on the left bank of the Dnieper.
The relationship of these variants with the epizootic situation was researched. For this purpose epizootological zoning of Ukraine according to the intensity of the epizootic situation in 2005–2009 was carried out. As a result of this analysis all the regions of Ukraine belong to three categories: high, medium and low epizootic situation intensity of rabies.
The projection of differentiated genetic clusters on the epizootic situation showed that cluster II circulating at Left Bank of the Dnieper in areas with high and medium intensity of the epizootic situation, and the cluster I – at the Right Bank of the Dnieper, mainly in the areas with low intensity of the epizootic situation.
That’s why our interest was in the degree of protection of rabies vaccines against street rabies virus isolates belonging to these two genetic clusters.
The commercial vaccines made with rabies virus vaccine strains SAD (Street-Alabama-Dufferin) and Wistar PM/WI were chosen to evaluate this parameter.
After the mathematical calculations of effective dose and the analysis of the data the less effective protection of rabies vaccines (at 29–30 %) against street rabies virus isolates belonging to cluster II in comparison with isolates belonging to cluster I irrespective to the strain vaccine is made was shown.
Conclusions
The data will be used for the effective planning of specific prophylaxis of rabies in Ukraine based on differentiated approach to distribution of rabies vaccines in according to region and their activity.
PMCID: PMC3692803
rabies vaccine; vaccine activity; street rabies virus isolates; genetic variants of rabies virus
20.  Intracerebral Administration of Recombinant Rabies Virus Expressing GM-CSF Prevents the Development of Rabies after Infection with Street Virus 
PLoS ONE  2011;6(9):e25414.
Recently it was found that prior immunization with recombinant rabies virus (RABV) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) (LBNSE-GM-CSF) resulted in high innate/adaptive immune responses and protection against challenge with virulent RABV (Wen et al., JVI, 2011). In this study, the ability of LBNSE-GM-CSF to prevent animals from developing rabies was investigated in mice after infection with lethal doses of street RABV. It was found that intracerebral administration of LBNSE-GM-CSF protected more mice from developing rabies than sham-treated mice as late as day 5 after infection with street RABV. Intracerebral administration of LBNSE-GM-CSF resulted in significantly higher levels of chemokine/cytokine expression and more infiltration of inflammatory and immune cells into the central nervous system (CNS) than sham-administration or administration with UV-inactivated LBNSE-GM-CSF. Enhancement of blood-brain barrier (BBB) permeability and increases in virus neutralizing antibodies (VNA) were also observed in mice treated with LBNSE-GM-CSF. On the other hand, intracerebral administration with UV-inactivated LBNSE-GM-CSF did not increase protection despite the fact that VNA were induced in the periphery. However, intracerebral administration with chemoattractant protein-1 (MCP-1, also termed CCL2) increased significantly the protective efficacy of UV-inactivated LBNSE-GM-CSF. Together these studies confirm that direct administration of LBNSE-GM-CSF can enhance the innate and adaptive immunity as well as the BBB permeability, thus allowing infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and prevent the development of rabies.
doi:10.1371/journal.pone.0025414
PMCID: PMC3182207  PMID: 21980450
21.  Antibody Quality and Protection from Lethal Ebola Virus Challenge in Nonhuman Primates Immunized with Rabies Virus Based Bivalent Vaccine 
PLoS Pathogens  2013;9(5):e1003389.
We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.
Author Summary
Ebola virus (EBOV) has been associated with outbreaks in human and nonhuman primate populations since 1976. With a fatality rate approaching 90%, EBOV is one of the most lethal infectious diseases in humans. The increased frequency of EBOV outbreaks along with its potential to be used as a bioterrorism agent has dramatically strengthened filovirus vaccine research and development. While there are currently no approved vaccines or post exposure treatments available for human use, several vaccine candidates have shown to protect nonhuman primates from lethal EBOV challenge. Our primary focus is to develop vaccine candidates to protect humans and endangered wildlife species at risk of infection in Africa. Here, we evaluated the efficacy and immunogenicity of our dual vaccines against EBOV and rabies virus (RABV) in rhesus macaques. Our live replication-competent vaccine provided 100% protection following EBOV challenge while the replication-deficient and inactivated candidates provided 50% protection. Interestingly, protection is dependent on the quality of the antibodies rather than the quantity. All three RABV-based EBOV vaccines did induce antibody levels necessary for protection from RABV infection. These results encourage the further development of these novel dual vaccines directed against two of the most lethal viral diseases.
doi:10.1371/journal.ppat.1003389
PMCID: PMC3667758  PMID: 23737747
22.  The use of an E1-deleted, replication-defective adenovirus recombinant expressing the rabies virus glycoprotein for early vaccination of mice against rabies virus. 
Journal of Virology  1997;71(5):3677-3683.
An E1-deleted, replication-defective adenovirus recombinant of the human strain 5 expressing the rabies virus glycoprotein, termed Adrab.gp, was tested in young mice. Mice immunized at birth with the Adrab.gp construct developed antibodies to rabies virus and cytokine-secreting lymphocytes and were protected against subsequent challenge. Maternal immunity to rabies virus strongly interferes with vaccination of the offspring with a traditional inactivated rabies virus vaccine. The immune response to the rabies virus glycoprotein, as presented by the Adrab.gp vaccine, on the other hand, was not impaired by maternal immunity. Even neonatal immunization of mice born to rabies virus-immune dams with Adrab.gp construct resulted in a long-lasting protective immune response to rabies virus, suggesting that this type of vaccine could be useful for immunization shortly after birth. Nevertheless, pups born to Adrab.gp virus-immune dams showed an impaired immune response to the rabies virus glycoprotein upon vaccination with the Adrab.gp virus, indicating that maternal immunity to the vaccine carrier affected the offspring's immune response to rabies virus.
PMCID: PMC191516  PMID: 9094641
23.  Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein. 
Journal of Virology  1992;66(5):2601-2604.
Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.
PMCID: PMC241012  PMID: 1560518
24.  Protection of dogs against death from experimental rabies by postexposure administration of rabies vaccine and hyperimmune globulin (human). 
Two experiments on simulated postexposure treatment were carried out in dogs using human rabies immunoglobulin (RIGH) and human diploid cell vaccine for human use. In one experiment, when animals were challenged by injecting street virus into the masseter muscle and treated with a combination of RIGH and vaccine, 50% of the animals were protected from rabies. In the other trial, in which animals were challenged by injecting the virus into the femoral muscle, treatment with RIGH and vaccine protected all the animals against rabies. To our knowledge this is the highest rate of postexposure survival in animals reported to date. In addition, five out of eight (62.5%) dogs that received RIGH alone after the virus challenge were protected, while none of the animals receiving vaccine alone were protected from rabies. These trials suggest that animals can be protected from rabies by postexposure treatment. The route of exposure and timing of the administration of vaccine and hyperimmune serum would seem to be important.
PMCID: PMC1255572  PMID: 2590870
25.  Immunization Against Rabies 
The methods used for both pre-exposure and post-exposure immunization against rabies were studied. In pre-exposure immunization duck embryo vaccine should be used. In post-exposure immunization either duck embryo or Semple-type vaccine appears to be effective in stimulating antibody production. Both vaccines may cause neurological sequelae. A dose of vaccine should be given 20-50 days after completion of the primary course of vaccination. Immune serum should be used in all severe exposures especially of the head and neck, and in individuals in whom the commencement of vaccination has been unduly delayed. In individuals who have been previously vaccinated reinforcing doses have been found to be effective even as long as 20 years after the primary vaccination. A tissue culture vaccine has been developed and is about to undergo field trials.
PMCID: PMC1935942  PMID: 6066820

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