Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus

Both rabies virus (RABV) and canine parvovirus (CPV) cause lethal diseases in dogs. In this study, both high egg passage Flury (HEP-Flury) strains of RABV and recombinant RABV carrying double RABV glycoprotein (G) gene were used to express the CPV virion protein 2 (VP2) gene, and were designated rHEP-VP2 and, rHEP-dG-VP2 respectively. The two recombinant RABVs maintained optimal virus titration according to their viral growth kinetics assay compared with the parental strain HEP-Flury. Western blotting indicated that G protein and VP2 were expressed in vitro. The expression of VP2 in Crandell feline kidney cells post-infection by rHEP-VP2 and rHEP-dG-VP2 was confirmed by indirect immunofluorescence assay with antibody against VP2. Immunogenicity of recombinant rabies viruses was tested in Kunming mice. Both rHEP-VP2 and rHEP-dG-VP2 induced high levels of rabies antibody compared with HEP-Flury. Mice immunized with rHEP-VP2 and rHEP-dG-VP2 both had a high level of antibodies against VP2, which can protect against CPV infection. A challenge experiment indicated that more than 80% mice immunized with recombinant RABVs survived after infection of challenge virus standard 24 (CVS-24). Together, this study showed that recombinant RABVs expressing VP2 induced protective immune responses to RABV and CPV. Therefore, rHEP-VP2 and rHEP-dG-VP2 might be potential combined vaccines for RABV and CPV.     

Recombinant canine distemper virus serves as bivalent live vaccine against rabies and canine distemper

Effective, safe, and affordable rabies vaccines are still being sought. Attenuated live vaccine has been widely used to protect carnivores from canine distemper. In this study, we generated a recombinant canine distemper virus (CDV) vaccine strain, rCDV-RVG, expressing the rabies virus glycoprotein (RVG) by using reverse genetics. The recombinant virus rCDV-RVG retained growth properties similar to those of vector CDV in Vero cell culture. Animal studies demonstrated that rCDV-RVG was safe in mice and dogs. Mice inoculated intracerebrally or intramuscularly with rCDV-RVG showed no apparent signs of disease and developed a strong rabies virus (RABV) neutralizing antibody response, which completely protected mice from challenge with a lethal dose of street virus. Canine studies showed that vaccination with rCDV-RVG induced strong and long-lasting virus neutralizing antibody responses to RABV and CDV. This is the first study demonstrating that recombinant CDV has the potential to serve as bivalent live vaccine against rabies and canine distemper in animals.     

Adult dogs receiving a rabies booster dose with a recombinant adenovirus expressing rabies virus glycoprotein develop high titers of neutralizing antibodies

Retired greyhound dogs, with low or absent antibody titers to rabies virus following previous vaccinations with commercially available vaccines, were immunized either subcutaneously or intramuscularly with a replication-defective recombinant adenovirus expressing the rabies virus glycoprotein termed Adrab.gp. Immunized animals developed high titers (geometric mean titers of 2630 and 5329) of viral neutralizing antibodies (VNA) against rabies virus by 10 days after vaccination. The antibody titers were even higher (geometric mean titers of 19349 and 122086) by 21 days after vaccination. The results indicate that the recombinant adenovirus expressing rabies virus glycoprotein is capable of inducing antibody immune responses in dogs and therefore may be developed as a rabies virus vaccine for dogs.     

Cross-protective and cross-reactive immune responses to recombinant vaccinia viruses expressing full-length lyssavirus glycoprotein genes

Lyssaviruses cause acute, progressive encephalitis in mammals. Current rabies vaccines offer protection against the lyssaviruses, with the notable exceptions of Mokola virus (MOKV), Lagos bat virus (LBV) and West Caucasian bat virus (WCBV). Here we describe the cross-protective and cross-reactive immune responses induced by experimental recombinant vaccinia viruses encoding the glycoprotein genes of rabies virus (RABV), MOKV and WCBV, either singly or in dual combinations. Constructs expressing a single glycoprotein gene protected mice against lethal intracranial challenge with homologous virus. Similarly, recombinants expressing glycoprotein genes from two different lyssaviruses offered mice protection against both homologous viruses. VNAb induced by vaccines that included a MOKV glycoprotein gene cross-neutralized LBV, but not WCBV. We concluded that a single recombinant poxvirus-vectored vaccine including MOKV and RABV glycoprotein genes, should be a major addition to available rabies biologics and should offer broad protection against all of the lyssaviruses, except WCBV.     

Addition of C3d-P28 adjuvant to a rabies DNA vaccine encoding the G5 linear epitope enhances the humoral immune response and confers protection

Rabies DNA vaccines based on full-length glycoprotein (G) induce virus neutralizing antibody (VNA) responses and protect against the virus challenge. Although conformational epitopes of G are the main target of VNAs, some studies have shown that a polypeptide linear epitope G5 is also able to induce VNAs. However, a G5 DNA vaccine has not been explored. While multiple doses of DNA vaccines are required in order to confer a protective immune response, this could be overcome by the inclusion of C3d-P28, a molecular adjuvant is know to improve the antibody response in several anti-viral vaccine models. To induce and enhance the immune response against rabies in mice, we evaluated two DNA vaccines based on the linear epitope G5 of Rabies Virus (RABV) glycoprotein (pVaxG5 vaccine) and another vaccine consisting of G5 fused to the molecular adjuvant C3d-P28 (pVaxF1 vaccine). VNA responses were measured in mice immunized with both vaccines. The VNA levels from the group immunized with pVaxG5 decreased gradually, while those from the group vaccinated with pVaxF1 remained high throughout the experimental study. After challenge with 22 LD50 of the Challenge Virus Strain (CVS), the survival rate of mice immunized with pVaxG5 and pVaxF1 was increased by 27% and 50% respectively, in comparison to the PBS group. Furthermore, the in vitro proliferation of anti-rabies specific spleen CD4+ and CD8+ T cells from mice immunized with pVaxF1 was observed. Collectively, these results suggest that the linear G5 epitope is a potential candidate vaccine. Furthermore, the addition of a C3d-P28 adjuvant contributed to enhanced protection, the sustained production of VNAs, and a specific T-cell proliferative response.     

Safety and serological response to a matrix gene-deleted rabies virus-based vaccine vector in dogs

Dogs account for the majority of human exposures and deaths due to rabies virus (RABV) worldwide. In this report, we show that a replication-deficient RABV-based vaccine in which the matrix gene is deleted (RABV-ΔM) is safe and induces rapid and potent VNA titers after a single inoculation in dogs. Average VNA titers peaked at 3.02 or 5.11 international units (IU/ml) by 14 days post-immunization with a single dose of 10⁶ or 10⁷ focus forming units (ffu), respectively, of RABV-ΔM. By day 70 post immunization, all dogs immunized with either dose of vaccine showed VNA titers >0.5 IU/ml, the level indicative of a satisfactory immunization. Importantly, no systemic or local reactions were noted in any dog immunized with RABV-ΔM. The elimination of dog rabies through mass vaccination is hindered by limited resources, requirement for repeat vaccinations often for the life of a dog, and in some parts of the world, inferior vaccine quality. Our preliminary safety and immunogenicity data in dogs suggest that RABV-ΔM might complement currently used inactivated RABV-based vaccines in vaccination campaigns by helping to obtain 100% response in vaccinated dogs, thereby increasing overall vaccination coverage.     

Virus neutralizing antibody response in mice and dogs with a bicistronic DNA vaccine encoding rabies virus glycoprotein and canine parvovirus VP2

A bicistronic DNA vaccine against rabies and parvovirus infection of dogs was developed by subcloning rabies glycoprotein and canine parvovirus (CPV) VP2 genes into a bicistronic vector. After characterizing the expression of both the proteins in vitro, the bicistronic DNA vaccine was injected in mice and induced immune response was compared with monocistronic DNA vaccines. There was no significant difference in ELISA and virus neutralizing (VN) antibody responses against rabies and CPV in mice immunized with either bicistronic or monocistronic DNA vaccine. Further, there was significantly similar protection in mice immunized with either bicistronic or monocistronic rabies DNA vaccine on rabies virus challenge. Similarly, dogs immunized with monocistronic and bicistronic DNA vaccines developed comparable VN antibodies against rabies and CPV. This study indicated that bicistronic DNA vaccine can be used in dogs to induce virus neutralizing immune responses against both rabies and CPV.     

High prevalence of antibodies against canine adenovirus (CAV) type 2 in domestic dog populations in South Africa precludes the use of CAV-based recombinant rabies vaccines

Rabies in dogs can be controlled through mass vaccination. Oral vaccination of domestic dogs would be useful in the developing world, where greater vaccination coverage is needed especially in inaccessible areas or places with large numbers of free-roaming dogs. From this perspective, recent research has focused on development of new recombinant vaccines that can be administered orally in a bait to be used as adjunct for parenteral vaccination. One such candidate, a recombinant canine adenovirus type 2 vaccine expressing the rabies virus glycoprotein (CAV2-RG), is considered a promising option for dogs, given host specificity and safety. To assess the potential use of this vaccine in domestic dog populations, we investigated the prevalence of antibodies against canine adenovirus type 2 in South African dogs. Blood was collected from 241 dogs from the Gauteng and KwaZulu-Natal provinces. Sampled dogs had not previously been vaccinated against canine adenovirus type 1 (CAV1) or canine adenovirus type 2 (CAV2). Animals from both provinces had a high percentage of seropositivity (45% and 62%), suggesting that CAV2 circulates extensively among domestic dog populations in South Africa. Given this finding, we evaluated the effect of pre-existing CAV-specific antibodies on the efficacy of the CAV2-RG vaccine delivered via the oral route in dogs. Purpose-bred Beagle dogs, which received prior vaccination against canine parvovirus, canine distemper virus and CAV, were immunized by oral administration of CAV2-RG. After rabies virus (RABV) infection all animals, except one vaccinated dog, developed rabies. This study demonstrated that pre-existing antibodies against CAV, such as naturally occurs in South African dogs, inhibits the development of neutralizing antibodies against RABV when immunized with a CAV-based rabies recombinant vaccine.     

HEP-Flury株病毒辅助质粒包装CTN株病毒基因组拯救狂犬病病毒

目的 以CTN株狂犬病病毒全长基因组cDNA重组质粒(pCTN-GFP)和HEP-Flury株病毒N、P、L辅助质粒共转染拯救具有活性的CTN株重组狂犬病病毒(CTN-GFP).方法 将CTN株狂犬病病毒全长基因组分4段进行扩增,体外连接扩增片段并克隆入表达载体中,构建CTN株狂犬病病毒全长基因组cDNA重组质粒,基因组ψ基因被标识基因GFP取代,通过与HEP-Flury株病毒N、P、L3个辅助质粒共转染BHK-21细胞,拯救能够稳定表达GFP的重组病毒CTN-GFP.结果 成功扩增全长基因组的4个基因片段,并将其克隆入表达载体,构建了CTN株狂犬病病毒全长基因组cDNA重组质粒pCTN-GFP,经酶切鉴定和序列测定证明pCTN-GFP为正确克隆,可直接用于病毒拯救.将pCTN-GFP与HEP-Flury株病毒辅助质粒共转染BHK-21细胞4d后,成功拯救出CTN株重组狂犬病病毒,重组病毒能够表达标识基因GFP,荧光显微镜下可直接观察到标识基因GFP的表达,设计跨GFP和L基因的特性引物对重组病毒进行RT-PCR检测,结果证明此病毒是从克隆的质粒中拯救的重组病毒.结论 HEP-Flury株病毒的结构蛋白能够包装并转录CTN株病毒的基因组RNA.     

Feline herpesvirus vectored-rabies vaccine in cats: A dual protection

In China, cats cause about 5% of human rabies cases. Rabies control in cats plays a role in achieving the ultimate goal of elimination of dog rabies-mediated human deaths. However, there is no cat-specific rabies vaccine in China yet. In this study, we constructed a recombinant rabies vaccine by using a felid herpesvirus 1 (FHV-1) isolate, and deleted the gI/E in the FHV-1 and replaced the region with a glycoprotein (G) of rabies virus (RABV) strain BD06 through homologous recombination. The recombinant virus FHV-RVG was recovered and purified, and the expression of RABV glycoprotein was verified by indirect immunofluorescent assay. For potency in cats, each animal was inoculated intranasally with 1?ml FHV-RVG at 10^6.5 TCID₅₀. Blood samples were collected at defined intervals for antibody titration. The animals were challenged by herpes and rabies after completion of vaccination on day 180 and day 194, respectively. Our results demonstrated all vaccinated cats generated antibodies against both FHV-1 and RABV, and reached an arbitrary protective titer?>?0.5?IU/ml for rabies viral neutralizing antibody (VNA) by day 14 post inoculation (dpi) and titer peaked on 30 dpi with VNA at 24.5?±?10.23?IU/ml. All vaccinated cats presented no clinical signs of FHV-1 infection and survived rabies challenge, while the control cats had severe rhinotracheitis and died from rabies after challenge. All this demonstrated that the FHV-based recombinant vaccine is effective in protection against both FHV-1 and RABV infections.     

Prime-boost immunization with DNA followed by a recombinant vaccinia virus expressing P50 induced protective immunity against Babesia gibsoni infection in dogs

A heterologous prime-boost immunization regime with priming DNA followed by recombinant vaccinia virus expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we constructed a recombinant plasmid and vaccinia virus, both of which expressed P50 of Babesia gibsoni, to investigate the immunogenicity and protective efficacy of a heterologous prime-boost immunization against canine babesiosis. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibody against P50 when compared with the control groups, and the antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. The prime-boost immunization regime induced a specific IgG2 antibody response and IFN-gamma production in dogs. Two weeks after the booster immunization with a recombinant vaccinia virus expressing P50, the dogs were challenged with B. gibsoni patasites. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia and a 2-day delay of the peak parasitemia. These results indicated that such a heterologous prime-boost immunization approach might be useful against B. gibsoni infection in dogs.     

Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein

We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Ebola virus (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.     

Poly(lactide-co-glycolide) microspheres: A potent oral delivery system to elicit systemic immune response against inactivated rabies virus

Rabies is an endemic, fatal zoonotic disease in the developing countries. Oral vaccination strategies are suitable for rabies control in developing countries. Studies were performed to investigate the suitability of poly(lactide-co-glycolide) (PLG) microspheres as an oral delivery system for β-propiolactone inactivated concentrated rabies virus (CRV). Immune responses induced by encapsulated (PLG + CRV) and un-encapsulated inactivated rabies virus after oral and intraperitoneal route administrations were compared. The anti-rabies virus IgG antibody titer, virus neutralizing antibody (VNA) titers obtained by mouse neutralization test (MNT) and IgG2a and IgG1 titers of mice group immunized orally with PLG + CRV showed significantly (p < 0.001) higher response than the group immunized orally with un-encapsulated CRV. There was no significant difference (p > 0.05) between groups inoculated by intraperitoneal route. The stimulation index (SI) obtained by lymphoproliferation assay of PLG + CRV oral group also showed significantly (p < 0.001) higher response than the group immunized orally with un-encapsulated CRV, suggesting that oral immunization activates Th1-mediated cellular immunity. Immunized mice of all experimental groups were challenged intracerebrally with a lethal dose of virulent rabies virus Challenge Virus Standard (CVS). The survival rates of mice immunized orally with PLG + CRV and CRV alone were 75% and 50%, respectively, whereas intraperitoneally immunized groups showed 100% protection. The overall results of humoral, cellular immune response and survival rates of mice immunized orally with PLG + CRV were significantly (p < 0.001) higher than those of mice immunized orally with CRV alone. These data suggest that the PLG encapsulated inactivated rabies virus can be used for oral immunization against rabies.     

Construction and immunogenicity of a recombinant fowlpox vaccine coexpressing S1 glycoprotein of infectious bronchitis virus and chicken IL-18

Infectious bronchitis virus (IBV) poses a major threat to the chicken industry worldwide. In this study, we developed a recombinant fowlpox virus (rFPV) vaccine expressing the IBV S1 gene and chicken interleukin-18 gene (IL-18), rFPV-S1/IL18. Recombinant plasmid pSY-S1/IL18 was constructed by cloning chicken IL-18 into fowlpox virus transfer plasmid containing S1 gene and transfected into the chicken embryo fibroblasts cell pre-infected with S-FPV-017 to generate rFPV-S1/IL18. Expression of the recombinant proteins was confirmed by RT-PCR and IFA. We also constructed the recombinant fowlpox virus rFPV-S1 without IL-18. One-day-old chickens were vaccinated by wing-web puncture with the two rFPVs, and the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels (P<0.05) elicited by either rFPV-S1 or rFPV-S1/IL18. The ratios of CD4(+) to CD8(+) in chickens immunized with rFPV-S1/IL18 were significantly higher (P<0.05) than in those immunized with rFPV-S1. All chickens immunized with rFPV-S1/IL18 were completely protected (20/20) after challenge with the virulent IBV HN99 strain 43 days after immunization, while only 15 out of 20 of the chickens immunized with the rFPV-S1 were protected. Our results show that the protective efficacy of the rFPV-S1 vaccine could be enhanced significantly by simultaneous expression of IL-18.     

Oral vaccination of dogs (Canis familiaris) with baits containing the recombinant rabies-canine adenovirus type-2 vaccine confers long-lasting immunity against rabies

Rabies is a reemerging and fatal infectious disease in Asia mainly caused by exposure to rabid dogs. Prevention of dog rabies would be the most effective way to stop rabies transmission to humans. However, vaccinating stray dogs in urban and rural areas using conventional vaccines is always difficult and is not cost-effective for use in most areas including China. Further to previous studies from our laboratory, we developed a bait containing the recombinant rabies vaccine and performed a non-parenteral trial in dogs. This vaccine was intranasally administrated once to 46 dogs in solution form with 1 x 10(8.5) PFU and orally to 90 dogs in specially designed baits with 3 x 10(8.5) PFU of the recombinant canine adenovirus. Results showed that about 87.5% (119/136) of the immunized dogs developed virus neutralizing antibodies (VNA). The immune response against rabies in dogs was detectable at 2-3 weeks after administration, reaching a peak by 5-6 weeks. Among the seroconverted animals, 90.8% (108/119) elicited a VNA response for over 24 months. The antibody titer during the 2 years was above 0.5IU /ml while showing a gradual but slow decline from the 6th week after vaccination. In a challenge experiment of 10 dogs with 60,000 mouse LD(50) of CVS-24 2 years after the vaccination, all the dogs survived. This demonstrated that the recombinant vaccine could be orally administrated and the bait was effective for the oral vaccination of dogs.     

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.     

Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep

Safe and efficient vaccination is important for rabies prevention in domestic animals. Replicative vectors expressing the rabies virus glycoprotein, derived from canine adenovirus have been reported to be promising vaccines in various animal models. In this paper we compare the potential of a replicative and a non-replicative vector, both based on canine adenovirus type 2 and expressing the rabies glycoprotein. Upon inoculation in sheep, immune responses against the rabies virus protein elicited by recombinant vectors were monitored. All immunised sheep produced a rapid and potent neutralizing antibody response against rabies virus after a single inoculation of either replicative or non-replicative recombinant canine adenovirus type 2. In addition, the non-replicative vector expressing the rabies glycoprotein stimulated antigen-specific CD4⁺ and CD8⁺ lymphocyte proliferation as well as IFN-γ production. These results suggest that vectors derived from canine adenovirus 2 could be considered for the development of promising vaccines in the ruminant species.     

Immunization of monkeys with rabies ribonucleoprotein (RNP) confers protective immunity against rabies

The ability of rabies virus ribonucleoprotein (RNP) to induce protective immunity against rabies and to prime for production of virus-neutralizing antibodies (VNA) was studied in monkeys. Following two immunizations with RNP, monkeys developed a strong anti-RNP response and were protected against a challenge infection with a lethal dose of street rabies virus. Monkeys that were primed with RNP and then immunized with a single dose of human diploid cell vaccine (HDCV) developed VNA titres comparable to the VNA titres in non-primed monkeys after a second HDCV immunization. The utility of rabies RNP for the pre-exposure prophylaxis of human rabies is discussed.     

A recombinant pseudorabies virus expressing rabies virus glycoprotein: safety and immunogenicity in dogs

Several recombinant vaccines expressing the rabies virus glycoprotein have been developed, particularly for the oral vaccination of wildlife. While these vaccines induce protective immunity in some animal species such as foxes, they are less effective in others. Pseudorabies virus (PRV) has been licensed for use as a live vaccine in pigs and possesses an excellent safety and efficacy record. We have used it to construct a recombinant virus, rPRV/eGFP/rgp, expressing the rabies virus glycoprotein. This recombinant virus has been shown to be safe for dogs by oral and intramuscular routes of inoculation and was demonstrated to induce immune responses against both pseudorabies and rabies in dogs after a single oral dose of 2 x 10(7.0) plaque forming units (PFU). Neutralizing antibody titers against rabies reached > 0.5 IU/ml and 1:64-1:128 against pseudorabies by 5 weeks post-vaccination in all dogs, indicating that the pseudorabies virus vector infected dogs and replicated in vivo, and that the rabies virus glycoprotein had been expressed and an effective immune response elicited. Antibody titers were maintained for over 6 months. This suggests that pseudorabies virus could be an effective live vector for recombinant rabies oral vaccination.