Virus-Like Vesicles Based on Semliki Forest Virus-Containing Rabies Virus Glycoprotein Make a Safe and Efficacious Rabies Vaccine Candidate in a Mouse Model.

Rabies is a fatal zoonosis that causes encephalitis in mammals, and vaccination is the most effective method to control and eliminate rabies. Virus-like vesicles (VLVs), which are characterized as infectious, self-propagating membrane-enveloped particles composed of only Semliki Forest virus (SFV) replicase and vesicular stomatitis virus glycoprotein (VSV-G), have been proven safe and efficient as vaccine candidates. However, previous studies showed that VLVs containing rabies virus glycoprotein (RABV-G) grew at relatively low titers in cells, impeding their potential use as a rabies vaccine. In this study, we constructed novel VLVs by transfection of a mutant SFV RNA replicon encoding RABV-G. We found that these VLVs could self-propagate efficiently in cell culture and could evolve to high titers (approximately 108 focus-forming units [FFU]/ml) by extensive passaging 25 times in BHK-21 cells. Furthermore, we found that the evolved amino acid changes in SFV nonstructural protein 1 (nsP1) at positions 470 and 482 was critical for this high-titer phenotype. Remarkably, VLVs could induce robust type I interferon (IFN) expression in BV2 cells and were highly sensitive to IFN-α. We found that direct inoculation of VLVs into the mouse brain caused reduced body weight loss, mortality, and neuroinflammation compared with the RABV vaccine strain. Finally, it could induce increased generation of germinal center (GC) B cells, plasma cells (PCs), and virus-neutralizing antibodies (VNAs), as well as provide protection against virulent RABV challenge in immunized mice. This study demonstrated that VLVs containing RABV-G could proliferate in cells and were highly evolvable, revealing the feasibility of developing an economic, safe, and efficacious rabies vaccine. IMPORTANCE VLVs have been shown to represent a more versatile and superior vaccine platform. In previous studies, VLVs containing the Semliki Forest virus replicase (SFV nsP1 to nsP4) and rabies virus glycoprotein (RABV-G) grew to relatively low titers in cells. In our study, we not only succeeded in generating VLVs that proliferate in cells and stably express RABV-G, but the VLVs that evolved grew to higher titers, reaching 108 FFU/ml. We also found that nucleic acid changes at positions 470 and 482 in nsP1 were vital for this high-titer phenotype. Moreover, the VLVs that evolved in our studies were highly attenuated in mice, induced potent immunity, and protected mice from lethal RABV infection. Collectively, our study showed that high titers of VLVs containing RABV-G were achieved, demonstrating that these VLVs could be an economical, safe, and efficacious rabies vaccine candidate.

Evaluation of an adjuvanted hydrogel-based pDNA nanoparticulate vaccine for rabies prevention and immunocontraception.

Immunocontraceptive vaccination is becoming an acceptable strategy in managing animal populations. Mass vaccination of dogs is the most cost-effective and efficient method to control rabies, and combination of rabies vaccination and animal population control will be an added advantage. In this study, we developed an adjuvanted hydrogel-based pDNA nanoparticulate vaccine for rabies protection and immunocontraception. In vivo, we observed an immune response skewed toward a Th2 type, in contrast to the Th1 type in our previous pDNA study. The observation was verified by the IgG2a/IgG1 ratio (<1), and cytokine expression profile of IL-4 and IFN-γ. The humoral immune response is key for rabies protection and a GnRH antibody-based immunocontraception. In mice, anti-GnRH antibody titers were detected 4 weeks after immunization and lasted for 12 weeks, post animal experiment was terminated. The adjuvanted pDNA nanoparticulate vaccine shows promise for future studies evaluating protection from rabies challenge and prevention of animal breeding.

[Rabies Virus Glycoprotein with a Consensus Amino Acid Sequence and a Lysosome Targeting Signal Causes Effective Production of Antibodies in DNA-Immunized Mice].

Safe and effective anti-rabies vaccines are intensely sought worldwide. DNA vaccines have already shown their efficacy and safety and have occupied a special place in the field. Two prototype anti-rabies DNA vaccines were compared for the potential to induce virus-specific antibody production. One vector contained a codon-optimized gene with a territory-adapted consensus sequence of the rabies virus glycoprotein. The other one expressed the same glycoprotein in fusion with a c-CD63 lysosome targeting motif at the C terminus. ELISA of serum samples from immunized mice showed that the c-CD63 variant induced more efficient antibody production and shifted the IgG2a/IgG1 ratio towards the Th2-type immune response. The results gave grounds to believe that the approach successfully applied to the rabies glycoprotein may help to develop new-generation anti-rabies vaccines.

Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine.

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.

Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: state of the science and planning the way forward.

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.

Modelling the challenges of managing free-ranging dog populations.

Free-ranging domestic dogs (FRD) are not only vectors of zoonoses of public health concern, but also pose direct threats to humans, livestock, and endangered wildlife. Many developing countries have struggled to control FRD, despite using both lethal and non-lethal methods. India has amongst the highest FRD populations globally and the highest incidences of dog-mediated human rabies, but only deploys Catch-Neuter-Vaccinate-Release (CNVR) for FRD control as a humane alternative to lethal methods, without evidence of it working successfully. Here, we use an agent-based dog population dynamics model to examine the time, effort, financial resources, and conditions needed to successfully control FRD in a typical urban setting. We simulate several scenarios, from an "ideal world" closed population with easily accessible dogs, to a more realistic open population with heterogeneity in catchability of dogs. In only one "best-case" scenario, CNVR resulted in a significant and lasting reduction in FRD, but with vaccination rates peaking only at 35%, which is half the WHO-recommended coverage. The customisable and portable modelling tool that we have developed allows managers to simulate real world processes and understand the expected effort needed to reduce regional dog populations, and assess methods for achieving effective anti-rabies vaccination coverage.

Local rabies transmission and regional spatial coupling in European foxes.

Infectious diseases are often transmitted through local interactions. Yet, both surveillance and control measures are implemented within administrative units. Capturing local transmission processes and spatial coupling between regions from aggregate level data is therefore a technical challenge that can shed light on both theoretical questions and practical decisions. Fox rabies has been eliminated from much of Europe through oral rabies vaccination (ORV) programmes. The European Union (EU) co-finances ORV to maintain rabies freedom in EU member and border states via a cordon sanitaire. Models to capture local transmission dynamics and spatial coupling have immediate application to the planning of these ORV campaigns and to other parts of the world considering oral vaccination. We fitted a hierarchical Bayesian state-space model to data on three decades of fox rabies cases and ORV campaigns from Eastern Germany. Specifically, we find that (i) combining regional spatial coupling and heterogeneous local transmission allows us to capture regional rabies dynamics; (ii) incursions from other regions account for less than 1% of cases, but allow for re-emergence of disease; (iii) herd immunity achieved through bi-annual vaccination campaigns is short-lived due to population turnover. Together, these findings highlight the need for regular and sustained vaccination efforts and our modelling approach can be used to provide strategic guidance for ORV delivery. Moreover, we show that biological understanding can be gained from inference from partially observed data on wildlife disease.

Induction of immune responses and protection in mice against rabies using a self-replicating RNA vaccine encoding rabies virus glycoprotein.

A self-replicating RNA vaccine encoding rabies virus glycoprotein gene was developed utilizing sindbis virus RNA replicon. The in vitro transcribed RNA (Sin-Rab-G RNA) was transfected in mammalian cells and analysed for self-replication and expression of rabies glycoprotein. To generate immune responses against rabies, mice were immunized with 10microg of Sin-Rab-G RNA and immune responses developed were compared with mice immunized with rabies DNA vaccine and commercial cell culture vaccine (Rabipur). The self-replicating rabies RNA vaccine generated cellular and humoral IgG responses similar to rabies DNA vaccine. On challenge with rabies virus CVS strain, rabies RNA vaccine conferred protection similar to rabies DNA vaccine. These results demonstrated that replicon-based self-replicating rabies RNA vaccine with 10microg dose was effective in inducing immune responses and protection similar to rabies DNA vaccine.

Evaluation of Different Stabilizers and Inactivating Compounds for the Enhancement of Vero Cell Rabies Vaccine Stability and Immunogenicity: In Vitro Study.

Inactivation of rabies virus is essential for rabies vaccine preparation where the inactivating compound that is currently recommended for rabies vaccine preparation is ß-propiolactone (ß-PL). This compound is considered better than phenol and formalin but it is expensive and potentially carcinogenic. Data revealed that Ascorbic acid (AA) with cupric ions could yield complete and irreversible inactivation of rabies virus without adversely affecting its antigenicity. Additionally, the results of testing the vaccine potency with the selected inactivating compounds were comparable (P<0.05), and ED50 was higher than the recommended World Health Organization (WHO) limits. The use of HemaGel (plasma substitute) for testing vaccine stabilization was compared with the currently used vaccine stabilizers (human albumin and lactose). HemaGel yielded better stability than the other tested stabilizers. Monitoring of cellular and humoral immune responses indicated that both the total IgG level against rabies vaccine and the IFN and IL5 levels obtained with the HemaGel-stabilized vaccines were higher than those obtained with human albumin- and lactose-stabilized vaccine candidates.

Benefit-cost analysis of the policy of mandatory annual rabies vaccination of domestic dogs in rabies-free Japan.

Japan is one of the few rabies-free countries/territories which implement the policy of mandatory vaccination of domestic dogs. In order to assess the economic efficiency of such policy in reducing the economic burden of a future canine rabies outbreak in Japan, a benefit-cost analysis (BCA) was performed using probabilistic decision tree modelling. Input data derived from simulation results of published mathematical model, field investigation conducted by the authors at prefectural governments, literature review, international or Japanese database and empirical data of rabies outbreaks in other countries/territories. The current study revealed that the annual costs of implementing the current vaccination policy would be US$160,472,075 (90% prediction interval [PI]: $149,268,935-171,669,974). The economic burden of a potential single canine rabies outbreak in Japan were estimated to be US$1,682,707 (90% PI: $1,180,289-2,249,283) under the current vaccination policy, while it would be US$5,019,093 (90% PI: $3,986,882-6,133,687) under hypothetical abolition of vaccination policy, which is 3-fold higher. Under a damage-avoided approach, the annual benefits of implementing the current vaccination policy in expected value were estimated to be US$85.75 (90% PI: $55.73-116.89). The benefit-cost ratio (BCR) was estimated to be 5.35 X 10(-7) (90% PI: 3.46 X 10(-7)-7.37 X 10(-7)), indicating that the implementation of the current policy is very economically inefficient for the purpose of reducing the economic burden of a potential canine rabies outbreak. In worse-case scenario analysis, the BCR would become above 1 (indicating economic efficiency) if the risk of rabies introduction increased to 0.04 corresponding to a level of risk where rabies would enter Japan in 26 years while the economic burden of a rabies outbreak under the abolition of vaccination policy increased to $7.53 billion. Best-case analysis further revealed that under relatively extreme circumstances the economic efficiency of the current policy could be improved by decreasing the vaccination price charged to dog owners, relaxing the frequency of vaccination to every two to three years and implementing the policy on a smaller scale, e.g. only in targeted prefectures instead of the whole Japan.

Pre-exposure rabies vaccination using purified chick embryo cell rabies vaccine intradermally is immunogenic and safe.

OBJECTIVE: To demonstrate the safety and immunogenicity of intradermal rabies pre-exposure prophylaxis with purified chick embryo cell vaccine (PCECV) in schoolchildren age 5 to 8 years in Thailand. STUDY DESIGN: In a randomized, open-label, phase II clinical trial, 2 or 3 intradermal doses of 0.1 mL PCECV (Rabipur) were administered to 703 schoolchildren on days 0 and 28 or on days 0, 7, and 28. In 206 children, 2 simulated post-exposure booster doses were given 1 year after the primary vaccination series. Rabies virus- neutralizing antibody (RVNA) titers were determined by the rapid fluorescent focus inhibition test. RESULTS: In school-age children in Thailand, a pre-exposure immunization regimen of 3 intradermal doses of PCECV produced adequate immune responses. After primary vaccination, all subjects developed RVNA titers > or =0.5 IU/mL and demonstrated a rapid increase in RVNA titer after 2 simulated post-exposure booster immunizations 1 year after the primary vaccination series. No serious adverse drug reactions occurred. CONCLUSIONS: Rabies pre-exposure immunization with PCECV is safe and immunogenic, and its implementation could save the lives of many children in rabies-endemic areas.

Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study.

Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

Enhanced Immune Response to Rabies Viruses by the Use of a Liposome Adjuvant in Vaccines.

Essen regimen, the Thai Red Cross two-site ID regimen, Zagreb schedule, and the eight-site ID regimen are the standard rabies vaccines recommended by the World Health Organization (WHO). In this study, a liposomal rabies vaccine (LipoRV) was developed, which was found to facilitate the production of rabies virus neutralizing antibody (RVNA) in BALB/c mice. Liposome solution was prepared with hydrogenated soya phosphatide and cholesterol. LipoRV composed of liposome solution and inactivated rabies vaccine (IRV). The immune responses were compared between the mice treated with either LipoRV or IRV. Higher levels of interleukin-2 (p < 0.05), interferon-γ (p < 0.01), and natural killer cell activity (p < 0.05) were observed in the mice immunized with LipoRV than those with IRV. The potency of LipoRV was significantly higher than that IRV (p < 0.05). In addition, three injections of LipoRV on days 0, 3, and 14 could elicit similar RVNA levels as the five shots of IRV. Our data also showed a higher survival rate in mice treated with three shots of LipoRV (56.2%) than five shots of IRV (40.6%). In conclusion, liposome enhances the immune response of mice to rabies vaccine and could be applied as a potential immunopotentiator.

Rabies in Europe--trends and developments.

Rabies continues to be a major public health concern both in Europe and worldwide. In the greater part of the European continent the disease has been eradicated, or its incidence drastically reduced, by oral vaccination of wildlife reservoirs. Yet, 'hot spots' still remain in north-eastern and south-eastern Europe with a high temporal variation of disease distribution in the previous decade. Rabies is predominantly transmitted by the red fox; but in recent years, especially in Eastern European countries, an independent infection cycle in raccoon dogs seems to have developed. Although countries such as the Czech Republic, Slovenia and Poland achieved very impressive rabies control results and trend analyses of rabies occurrence showed a long- and short-term decrease, there is still need for improved rabies surveillance and control in Europe. The Rabies Bulletin Europe (RBE), a quarterly, printed and on-line publication, aims to improve this situation. Besides the need to improve the quality of data in some countries, it will provide a public domain rabies database in the near future. Further strategic cooperation is necessary to control the disease in areas of European public health concern.

Elimination of rabies in the Czech Republic.

Rabies in foxes was widespread in the territory of the Czech Republic after the Second World War. The first three cases of fox rabies appeared in North and South Bohemia in 1947. In the following year, a considerable increase in rabies incidence was reported and of the 146 cases, 106 (74 %) of the 117 wildlife cases were in foxes. Rabies in foxes and other wild animals was verified in many border Czech and Moravian districts. Sylvatic rabies was established in the Czech Republic and persisted there enzootically for many years as a continuing problem. The red fox (Vulpes vulpes) became the principal vector and reservoir. Averaging rabies cases over the long term, foxes account for about 90 % of all positive cases. The highest incidence of rabies was recorded in 1984, reaching 2,232 reported cases. Since then a continual decline in the number of cases has been visible, especially since 1992 when the positive effect of oral vaccination became evident.

First oral vaccination of wildlife against rabies in Estonia.

Estonia is a small country, with many islands, that covers a total area of 45,000 km2 in the Baltic region. Rabies is widely distributed all over the country, even on some islands. The disease is maintained by two species: red foxes and raccoon dogs. In 2003, 813 cases of rabies were recorded with 315 cases in foxes and 362 cases in raccoon dogs; since 2002 the latter species has had the highest level of rabies infection among all animal species in Estonia. The measure used to control rabies is vaccination of dogs and cats, as they are the source on infection of humans. In Spring 2004, a limited trial of oral vaccination of wildlife was conducted. The goal of this was to train the actors who will be implicated in larger future campaigns. This trial was conducted on Vormsi island (92 km2), located 10 km away from the west coast. Oral vaccination was performed manually over three days with SAG2 baits (Virbac company, France), by three teams.

Determination of efficacy of rabies vaccine in the Saratov region.

The most complicated situation concerning rabies is seen in the Saratov region. The basic reservoir of rabies in this region is wild carnivores. A study was conduted in three districts of the Saratov region to define the efficiency of measures taken with the "Sinrab" rabies vaccine containing "RV-97" used for oral immunisation of wild animals. As a result of the experiment it was established that specific prevention of rabies in wildlife in enzootic territories resulted in a decrease of rabies cases.

Means used for terrestrial rabies elimination in France and policy for rabies surveillance in case of re-emergence.

In France, the first case of wildlife rabies was detected in 1968, with the red fox (Vulpes vulpes) as reservoir and vector of terrestrial rabies. The last case was reported at the end of 1998. The maximum infected area amounted to 140,000 km2 in 1989 with a record number of 4,213 infected wild and domestic animals. The contaminated areas included various landscapes such as low populated farming areas and low mountainous areas as well as very densely populated areas and industrial areas contiguously urbanised. Oral vaccination of wildlife against rabies was implemented in 1986 and 1987 on limited areas treated by hand distribution and then helicopter distribution was implemented in 1988. The control of wildlife rabies was centralised at the country level in one institute for elaboration of baiting strategies, rabies surveillance network, laboratory investigations such as tetracycline and serological testing, vaccine titration and rabies diagnosis. Oral vaccination campaigns were organised in spring and in autumn by dropping annually 40 baits/km2. The following vaccine baits were used: SAD B19 from 1986 to 1992, SAG1/SAG2 from 1990 to 2003 and VRG from 1989 to 2003. A cost analysis study of the system of oral vaccination implemented in France demonstrated that it is beneficial compared to the traditional expenses of rabies control. Animal rabies prophylaxis has been progressively adapted to the disease free status of the country; the surveillance network for rabies is still working and an emergency procedure is in place in the event of a re-emergence of the disease in the context of high density fox populations.

Epidemiological analysis of setbacks in oral vaccination in the final stage of fox rabies elimination in densely populated areas in Germany.

Despite a long history of oral vaccination of foxes (OVF) against rabies, in a few restricted areas of Germany rabies is still endemic, posing a continuous risk of re-introduction of rabies into adjacent, rabies-free areas. The endemic area is characterized by a high density population. It is hypothesized that the degree of urbanization in the area under consideration influences the number of rabies cases via the mode of bait distribution. In urban areas vaccine baits are distributed by hand, whereas in non-urban areas baits can be distributed aerially with the help of fixed-winged aircraft. Statistical analysis of the effect of the mode of bait distribution upon the number of rabies cases shows a significant influence. In areas where baits are distributed by hand the number of rabies cases is significantly higher than the expected number. This finding forces managers to reassess the procedure of bait distribution by hand in urban areas, taking into account the ecologically and biologically different dynamics of urban fox populations. If the oral vaccination of foxes in urban areas can be refined, rabies eradication in Germany is expected to succeed in due course.

Rabies vaccination programme for red foxes (Vulpes vulpes) and golden jackals (Canis aureus) in Israel (1999-2004).

Since 1956, red foxes (Vulpes vulpes) and, to a lesser extent, golden jackals (Canis aureus), have been the primary vectors maintaining endemic wildlife rabies in Israel. Starting in the autumn of 1998, oral rabies vaccination campaigns have been conducted in Israel targeting these two wildlife species, with increasing yearly geographical extension. Significant data have been accumulated from an area of approximately 5,200 km2 in Northern Israel. In the spring of 2003 the project was extended to 14,000 km2 and in the autumn to 21,000 km2, covering almost all inhabited areas in Israel and the West Bank. A total of two million RABORAL V-RG (Merial) vaccine-filled baits were distributed bi-annually by plane or helicopter at 14-19 baits km2. Since the onset of oral vaccination activities in 1998, annual bait acceptance in the vaccination zones has been demonstrated by biomarker detection (with tetracycline) in 55 % (429/783) of bone samples of target animals submitted for diagnosis. In 1999 to 2004, vaccine contact and induction of immunity in animals collected from the vaccination zones were reflected by seroconversion in 66 of 284 animals (23 %). By the year 2004, rabies cases declined sharply in all progressively vaccinated areas.