The Phylogeography of Rabies in Grenada,West Indies,and Implications for Control

In Grenada, West Indies, rabies is endemic, and is thought to be maintained in a wildlife host, the small Indian mongoose (Herpestes auropunctatus) with occasional spillover into other hosts. Therefore, the present study was undertaken to improve understanding of rabies epidemiology in Grenada and to inform rabies control policy. Mongooses were trapped island-wide between April 2011 and March 2013 and examined for the presence of Rabies virus (RABV) antigen using the direct fluorescent antibody test (dFAT) and PCR, and for serum neutralizing antibodies (SNA) using the fluorescent antibody virus neutralization test (FAVN). An additional cohort of brain samples from clinical rabies suspects submitted between April 2011 and March 2014 were also investigated for the presence of virus. Two of the 171 (1.7%) live-trapped mongooses were RABV positive by FAT and PCR, and 20 (11.7%) had SNAs. Rabies was diagnosed in 31 of the submitted animals with suspicious clinical signs: 16 mongooses, 12 dogs, 2 cats and 1 goat. Our investigation has revealed that rabies infection spread from the northeast to the southwest of Grenada within the study period. Phylogenetic analysis revealed that the viruses from Grenada formed a monophyletic clade within the cosmopolitan lineage with a common ancestor predicted to have occurred recently (6–23 years ago), and are distinct from those found in Cuba and Puerto Rico, where mongoose rabies is also endemic. These data suggest that it is likely that this specific strain of RABV was imported from European regions rather than the Americas. These data contribute essential information for any potential rabies control program in Grenada and demonstrate the importance of a sound evidence base for planning interventions.     

Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans.     

Bat Rabies in Guatemala

Rabies in bats is considered enzootic throughout the New World, but few comparative data are available for most countries in the region. As part of a larger pathogen detection program, enhanced bat rabies surveillance was conducted in Guatemala, between 2009 and 2011. A total of 672 bats of 31 species were sampled and tested for rabies. The prevalence of rabies virus (RABV) detection among all collected bats was low (0.3%). Viral antigens were detected and infectious virus was isolated from the brains of two common vampire bats (Desmodus rotundus). RABV was also isolated from oral swabs, lungs and kidneys of both bats, whereas viral RNA was detected in all of the tissues examined by hemi-nested RT-PCR except for the liver of one bat. Sequencing of the nucleoprotein gene showed that both viruses were 100% identical, whereas sequencing of the glycoprotein gene revealed one non-synonymous substitution (302_T,S). The two vampire bat RABV isolates in this study were phylogenetically related to viruses associated with vampire bats in the eastern states of Mexico and El Salvador. Additionally, 7% of sera collected from 398 bats demonstrated RABV neutralizing antibody. The proportion of seropositive bats varied significantly across trophic guilds, suggestive of complex intraspecific compartmentalization of RABV perpetuation.     

Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein

Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes.     

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

Rabies virus (RABV) induces encephalomyelitis in humans and animals. One of the major problems with rabies is that the infected individuals most often do not develop virus neutralizing antibodies (VNA). In this study we have investigated the host immune response to RABV infection in dogs, using a live-attenuated (TriGAS) or a wild-type (wt) (DRV-NG11) RABV isolated from a rabid dog.

Methodology/Principal Findings

The experimental infection of dogs with TriGAS induced high levels of VNA in the serum, whereas wt RABV infection did not. Dogs infected with TriGAS developed antibodies against the virus including its glycoprotein, whereas dogs infected with DRV-NG11 only developed rabies antibodies that are presumably specific for the nucleoprotein, (N) and not the glycoprotein (G). We show that infection with TriGAS induces early activation of B cells in the draining lymph nodes and persistent activation of DCs and B cells in the blood. On the other hand, infection with DRV-NG11 fails to induce the activation of DCs and B cells and further reduces CD4 T cell production. Further, we show that intrathecal (IT) immunization of TriGAS not only induced high levels of VNA in the serum but also in the CSF while intramuscular (IM) immunization of TriGAS induced VNA only in the serum. In addition, high levels of total protein and WBC were detected in the CSF of IT immunized dogs, indicating the transient enhancement of blood-brain barrier (BBB) permeability, which is relevant to the passage of immune effectors from periphery into the CNS.

Conclusions/Significance

IM infection of dogs with TriGAS induced the production of serum VNA whereas, IT immunization of TriGAS in dogs induces high levels of VNA in the periphery as well as in the CSF and transiently enhances BBB permeability. In contrast, infection with wt DRV-NG11 resulted in the production of RABV-reactive antibodies but VNA and antibodies specific for G were absent. As a consequence, all of the dogs infected with wt DRV-NG11 succumbed to rabies. Thus the failure to activate protective immunity is one of the important features of RABV pathogenesis in dogs.     

λ-Carrageenan P32 Is a Potent Inhibitor of Rabies Virus Infection

Rabies, caused by rabies virus (RABV), is an acute, fatal encephalitic disease that affects many warm-blooded mammals. Currently, post-exposure prophylaxis regimens are effective for most rabies cases, but once the clinical signs of the disease appear, current treatment options become ineffective. Carrageenan has been reported as a potent inhibitor of many viruses. In this study, the λ-carrageenan (λ-CG) P32 was investigated for its potential role in inhibiting RABV infection. Our results show that P32 specifically inhibits the replication of several RABV strains but not vesicular stomatitis virus in multiple cell lines and shows low cytotoxicity. P32 mainly abrogated viral replication during the early stage of the post-adsorption period. Further studies demonstrated that P32 could affect not only viral internalization but also viral uncoating by blocking cell fusion mediated by RABV glycoprotein. Moreover, P32 can fully inhibit RABV infection in vitro during the post-adsorption period, whereas heparin and heparan sulfate, which possess similar structures to P32, showed significant but not complete inhibition of RABV infectivity. Collectively, our results indicate that λ-CG P32 is a promising agent that can inhibit RABV infection mainly by inhibiting viral internalization and glycoprotein-mediated cell fusion and can be used for the development of novel anti-RABV drugs.     

Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein

The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.     

Recombinant monoclonal antibodies for rabies post-exposure prophylaxis

Rabies virus is a prototypical neurotropic virus that causes one of the most dangerous zoonotic diseases in humans. Humanized or fully human monoclonal antibodies (mAb) that neutralize rabies virus would be the basis for powerful post-exposure prophylaxis of rabies in humans, having several significant benefits in comparison with human or equine rabies polyclonal immunoglobulins. The most advanced antibodies should broadly neutralize natural rabies virus isolates, bind with conserved antigenic determinants of the rabies virus glycoprotein, and show high neutralizing potency in assays in vivo. The antibodies should recognize nonoverlapping epitopes if they are used in combination. This review focuses on basic requirements for anti-rabies therapeutic antibodies. The urgency in the search for novel rabies post-exposure prophylaxis and methods of development of anti-rabies human mAb cocktail are discussed. The rabies virus structure and pathways of its penetration into the nervous system are also briefly described.     

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.     

Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris

In this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV‐G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV‐G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV‐G gene was not tremendously increased as compared with the non‐optimized one. In addition, similar levels of RABV‐G were obtained when α‐factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high‐level expression of RABV‐G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV‐G expression cassette indicating that folding was the limiting step for RABV‐G secretion. To circumvent this limitation, co‐overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV‐G reached 1230 ng ml^?1. Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.     

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 T_fh 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.     

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.     

High diversity of rabies viruses associated with insectivorous bats in Argentina: presence of several independent enzootics

Background

Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory.

Methods

We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008.

Results

Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples.

Conclusions

This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection.     

Enhanced transport of plant‐produced rabies single‐chain antibody‐RVG peptide fusion protein across an in cellulo blood–brain barrier device

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood–brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single‐chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv‐RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv‐RVG fusion. Both ScFv and ScFv‐RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv‐RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant‐produced ScFv‐RVG^P fusion could translocate across the cells. This study indicates that the plant‐produced ScFv‐RVG^P fusion protein was able to cross the in celluloBBB and neutralize RABV.     

A Novel Rabies Vaccine Based on a Recombinant Parainfluenza Virus 5 Expressing Rabies Virus Glycoprotein

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.     

The influence of homologous vs. heterologous challenge virus strains on the serological test results of rabies virus neutralizing assays

The effect that the relatedness of the viral seed strain used to produce rabies vaccines has to the strain of challenge virus used to measure rabies virus neutralizing antibodies after vaccination was evaluated. Serum samples from 173 subjects vaccinated with either purified Vero cell rabies vaccine (PVRV), produced from the Pittman Moore (PM) seed strain of rabies virus, or purified chick embryo cell rabies vaccine (PCECV), produced from the Flury low egg passage (Flury-LEP) seed strain of rabies virus, were tested in parallel assays by RFFIT using a homologous and a heterologous testing system. In the homologous system, CVS-11 was used as the challenge virus in the assay to evaluate the humoral immune response in subjects vaccinated with PVRV and Flury-LEP was used for subjects vaccinated with PCECV. In the heterologous system, CVS-11 was used as the challenge virus in the assay to evaluate subjects vaccinated with PCECV and Flury-LEP was used for subjects vaccinated with PVRV. Although the difference in G protein homology between the CVS-11 and Flury-LEP rabies virus strains has been reported to be only 5.8%, the use of a homologous testing system resulted in approximately 30% higher titers for nearly two-thirds of the samples from both vaccine groups compared to a heterologous testing system. The evaluation of equivalence of the immune response after vaccination with the two different vaccines was dependent upon the type of testing system, homologous or heterologous, used to evaluate the level of rabies virus neutralizing antibodies. Equivalence between the vaccines was achieved when a homologous testing system was used but not when a heterologous testing system was used. The results of this study indicate that the strain of virus used in the biological assays to measure the level of rabies virus neutralizing antibodies after vaccination could profoundly influence the evaluation of rabies vaccines.     

Biotechnology advances: A perspective on the diagnosis and research of Rabies Virus

Rabies is a widespread zoonotic disease responsible for approximately 55,000 human deaths/year. The direct fluorescent antibody test (DFAT) and the mouse inoculation test (MIT) used for rabies diagnosis, have high sensitivity and specificity, but are expensive and time-consuming. These disadvantages and the identification of new strains of the virus encourage the use of new techniques that are rapid, sensitive, specific and economical for the detection and research of the Rabies Virus (RABV). Real-time RT-PCR, phylogeographic analysis, proteomic assays and DNA recombinant technology have been used in research laboratories. Together, these techniques are effective on samples with low virus titers in the study of molecular epidemiology or in the identification of new disease markers, thus improving the performance of biological assays. In this context, modern advances in molecular technology are now beginning to complement more traditional approaches and promise to revolutionize the diagnosis of rabies. This brief review presents some of the recent molecular tools used for RABV analysis, with emphasis on rabies diagnosis and research.     

Presence of Virus Neutralizing Antibodies in Cerebral Spinal Fluid Correlates with Non-Lethal Rabies in Dogs

Background

Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans.

Methodology/Principal Findings

Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS.

Conclusions/Significance

Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection.     

Prevention of rabies virus infection in dogs by a recombinant canine adenovirus type-2 encoding the rabies virus glycoprotein

Safe and effective vaccination is important for rabies prevention in animals. Although several genetically engineered rabies vaccines have been developed, few have been licensed for use, principally due to biosafety concerns or due to poor efficacy in animal models. In this paper, we describe the construction and characterization of a replication-competent recombinant canine adenovirus type-2 expressing the rabies virus glycoprotein (SRV9 strain) by a different strategy from that reported previously, i.e., the recombinant genome carrying the glycoprotein cDNA was generated by a series of strictly gene cloning steps, infectious recombinant virus was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. This recombinant virus, CAV-E3delta-CGS, was subcutaneously injected into dogs. All vaccinated dogs produced effective neutralizing antibodies after one inoculation and a stronger anamnestic immune response was produced after booster injection. The immunized dogs could survive the challenge of 60,000 mouse LD50 CVS-24, which is lethal to all unimmunized dogs and is comparable to the conventional vaccines. The immunity lasts for months with a protective level of neutralizing antibody. This recombinant virus would be an alternative to the attenuated and the inactivated rabies vaccines and be prospective in immunizing dogs against rabies.     

In vitro rabies vaccine potency appraisal by ELISA: Advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody

The replacement of the in vivo potency test (NIH test) for rabies vaccine evaluation by in vitro methods is at present discussed in many reports and also by WHO expert working groups. For this purpose, in vitro glycoprotein titration has been proposed. Among the different glycoprotein assays, we have studied two ELISA methods (immunocapture and direct plate coating with the antigen to be tested) using neutralizing mono- and polyclonal antibodies. In our view, the immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.