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.     

Strategic Focus on 3R Principles Reveals Major Reductions in the Use of Animals in Pharmaceutical Toxicity Testing

The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level.     

Cost-Effectiveness Analysis of Chemical Testing for Decision-Support: How to Include Animal Welfare?

Toxicity testing for regulatory purposes raises the question of test selection for a particular endpoint. Given the public's concern for animal welfare, test selection is a multi-objective decision problem that requires balancing information outcome, animal welfare loss, and monetary testing costs. This paper demonstrates the applicability of cost-effectiveness analysis as a decision-support tool for test selection in a regulatory context such as, for example, the new European chemicals legislation (REACH). We distinguish different decision-making perspectives, in particular the regulator's and chemical industry's perspectives, and we discuss how cost-effectiveness analysis can be applied to test selection from both perspectives. Furthermore, we show how animal welfare goals can be included in cost-effectiveness analyses, and we provide a three-dimensional extension to the standard cost-effectiveness analysis if animal welfare loss cannot be valued in monetary terms. To illustrate how cost-effectiveness analysis works in different settings, we apply our model to a simple case of selecting short-term tests for mutagenicity. We demonstrate that including sufficiently high values for animal welfare induces cost-effective replacements of animal testing. Furthermore, we show that the regulator and chemical companies face different tradeoffs when selecting tests. This may lead to different choices of tests or testing systems.     

A One Health Framework for the Evaluation of Rabies Control Programmes: A Case Study from Colombo City,Sri Lanka

Background

One Health addresses complex challenges to promote the health of all species and the environment by integrating relevant sciences at systems level. Its application to zoonotic diseases is recommended, but few coherent frameworks exist that combine approaches from multiple disciplines. Rabies requires an interdisciplinary approach for effective and efficient management.

Methodology/Principal Findings

A framework is proposed to assess the value of rabies interventions holistically. The economic assessment compares additional monetary and non-monetary costs and benefits of an intervention taking into account epidemiological, animal welfare, societal impact and cost data. It is complemented by an ethical assessment. The framework is applied to Colombo City, Sri Lanka, where modified dog rabies intervention measures were implemented in 2007. The two options included for analysis were the control measures in place until 2006 (“baseline scenario”) and the new comprehensive intervention measures (“intervention”) for a four-year duration. Differences in control cost; monetary human health costs after exposure; Disability-Adjusted Life Years (DALYs) lost due to human rabies deaths and the psychological burden following a bite; negative impact on animal welfare; epidemiological indicators; social acceptance of dogs; and ethical considerations were estimated using a mixed method approach including primary and secondary data. Over the four years analysed, the intervention cost US $1.03 million more than the baseline scenario in 2011 prices (adjusted for inflation) and caused a reduction in dog rabies cases; 738 DALYs averted; an increase in acceptability among non-dog owners; a perception of positive changes in society including a decrease in the number of roaming dogs; and a net reduction in the impact on animal welfare from intermediate-high to low-intermediate.

Conclusions

The findings illustrate the multiple outcomes relevant to stakeholders and allow greater understanding of the value of the implemented rabies control measures, thereby providing a solid foundation for informed decision-making and sustainable control.     

Attitudes toward Animals of Students at Three European Veterinary Medicine Schools in Italy and Spain

The European Veterinary Code of Conduct recognizes the crucial role of veterinarians in improving animal welfare and maintaining its standards. However, several studies have claimed that veterinary students’ attitudes toward animals may worsen as they progress through their academic training. This study aimed to investigate students’ attitudes toward nonhuman animal use in three European veterinary medicine schools (Italy and Spain). For this purpose, 565 veterinary students completed a questionnaire consisting of a range of items grouped into four animal-use categories: “Research,” “Entertainment,” “Utilitarianism,” and “Veterinary Issues.” Items were scored on a 5-point Likert-type scale, with higher scores indicative of higher concern regarding animal welfare. Results showed that the use of animals for “Entertainment” and questions related to “Veterinary Issues” raised the most concern among the veterinary students, while the use of animals for “Research” was of least concern. Moreover, we also examined some potentially confounding factors: age, academic year (first to fifth), gender, previous experience with pets, and university. Female students had a higher concern for animal welfare compared with their male counterparts. Students in their earlier stages of training as veterinarians also exhibited a greater concern for animal welfare compared with those of later academic years. Other factors affecting students’ attitudes toward animal use were the age of the students and the university they were enrolled at. The findings of this study confirm that attitudes toward animal use are not homogeneous and are associated with students’ demographic, educational, and personal characteristics.     

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.     

In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife

Although rabies incidence has fallen sharply over the past decades in Europe, the disease is still present in Eastern Europe. Oral rabies immunization of wild animal rabies has been shown to be the most effective method for the control and elimination of rabies. All rabies vaccines used in Europe are modified live virus vaccines based on the Street Alabama Dufferin (SAD) strain isolated from a naturally-infected dog in 1935. Because of the potential safety risk of a live virus which could revert to virulence, the genetic composition of three commercial attenuated live rabies vaccines was investigated in two independent laboratories using next genome sequencing. This study is the first one reporting on the diversity of variants in oral rabies vaccines as well as the presence of a mix of at least two different variants in all tested batches. The results demonstrate the need for vaccine producers to use new robust methodologies in the context of their routine vaccine quality controls prior to market release.     

Potency estimation of vaccine batches remains unaffected by anesthesia for intracerebral injection

Potency testing of rabies and whole-cell pertussis vaccine batches is still performed by an intracerebral (i.c.) challenge test, in conformity with international regulatory requirements. For the i.c. injection, the use of anesthesia is strongly recommended to alleviate the severe pain induced by the procedure. Today, anesthesia is not consistently mentioned in regulatory requirements, in contrast to the times when the potency tests were developed. The introduction of anesthesia is hampered, due to the lack of data on a hypothetical impact of anesthesia on potency estimation. Here, we show the comparative analysis of the extensive batch release data set of a rabies vaccine for human use that was tested in two laboratories of which only one applied anesthesia. In essence, we find that the mean batch test results were similar to each other, demonstrating that anesthesia for i.c. injection does not interfere with potency estimation. Consequently, we recommend the update of regulatory requirements and protocols and support the implementation of anesthesia for i.c. injection.     

Current characteristics of animal rabies cases in Thailand and relevant risk factors identified by a spatial modeling approach

The situation of human rabies in Thailand has gradually declined over the past four decades. However, the number of animal rabies cases has slightly increased in the last ten years. This study thus aimed to describe the characteristics of animal rabies between 2017 and 2018 in Thailand in which the prevalence was fairly high and to quantify the association between monthly rabies occurrences and explainable variables using the generalized additive models (GAMs) to predict the spatial risk areas for rabies spread. Our results indicate that the majority of animals affected by rabies in Thailand are dogs. Most of the affected dogs were owned, free or semi-free roaming, and unvaccinated. Clusters of rabies were highly distributed in the northeast, followed by the central and the south of the country. Temporally, the number of cases gradually increased after June and reached a peak in January. Based on our spatial models, human and cattle population density as well as the spatio-temporal history of rabies occurrences, and the distances from the cases to the secondary roads and country borders are identified as the risk factors. Our predictive maps are applicable for strengthening the surveillance system in high-risk areas. Nevertheless, the identified risk factors should be rigorously considered and integrated into the strategic plans for the prevention and control of animal rabies in Thailand.     

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.     

Epidemiology,Impact and Control of Rabies in Nepal: A Systematic Review

Background

Rabies is a vaccine-preventable viral zoonosis belonging to the group of neglected tropical diseases. Exposure to a rabid animal may result in a fatal acute encephalitis if effective post-exposure prophylaxis is not provided. Rabies occurs worldwide, but its burden is disproportionately high in developing countries, including Nepal. We aimed to summarize current knowledge on the epidemiology, impact and control of rabies in Nepal.

Methods

We performed a systematic review of international and national scientific literature and searched grey literature through the World Health Organization Digital Library and the library of the National Zoonoses and Food Hygiene Research Centre, Nepal, and through searching Google and Google Scholar. Further data on animal and human rabies were obtained from the relevant Nepalese government agencies. Finally, we surveyed the archives of a Nepalese daily to obtain qualitative information on rabies in Nepal.

Findings

So far, only little original research has been conducted on the epidemiology and impact of rabies in Nepal. Per year, rabies is reported to kill about 100 livestock and 10–100 humans, while about 1,000 livestock and 35,000 humans are reported to receive rabies post-exposure prophylaxis. However, these estimates are very likely to be serious underestimations of the true rabies burden. Significant progress has been made in the production of cell culture-based anti-rabies vaccine and rabies immunoglobulin, but availability and supply remain a matter of concern, especially in remote areas. Different state and non-state actors have initiated rabies control activities over the years, but efforts typically remained focalized, of short duration and not harmonized. Communication and coordination between veterinary and human health authorities is limited at present, further complicating rabies control in Nepal. Important research gaps include the reporting biases for both human and animal rabies, the ecology of stray dog populations and the true contribution of the sylvatic cycle.

Interpretation

Better data are needed to unravel the true burden of animal and human rabies. More collaboration, both within the country and within the region, is needed to control rabies. To achieve these goals, high level political commitment is essential. We therefore propose to make rabies the model zoonosis for successful control in Nepal.     

The effect of strain differences on the assay of rabies virus glycoprotein by single radial immunodiffusion

Antigenic differences between rabies virus strains used for vaccine manufacture can be demonstrated using monoclonal antibodies. We have shown that these differences are sufficiently large to affect the potency values of vaccines measured in single radial immunodiffusion (SRD) assays if the reference and test vaccines are antigenically heterologous. The production of reagents for use in SRD assays for each strain of rabies virus should be considered.     

Effects of Natural Barriers and Habitat on the Western Spread of Raccoon Rabies in Alabama

Abstract: Although domestic animal transmission of rabies has largely been mitigated, the disease remains a concern in both Europe and North America where wildlife transmission has caused epizootics. Raccoon (Procyon lotor) rabies was established in Alabama, USA, in 1975, primarily in the southeastern corner of the state. However, with the exception of isolated events, rabies has not continued to spread westward across the Alabama River. We monitored movements of 100 radiocollared raccoons on 2 sites within hardwood and agriculture habitats in a rabies enzootic area east of the Alabama River, in managed pine habitat area west of the river where rabies sporadically occurs, and in a mixed pine hardwood area outside of the known rabies enzootic area to determine if raccoon movements and habitat use in certain habitat types and the presence of a river may serve as natural barriers preventing the western spread of rabies in Alabama. We also examined raccoon contact rates to determine if they influence disease transmission through static and dynamic interactions. Raccoons in mixed pine-hardwood forest habitats had smaller home ranges and less overlap of ranges compared to the other 3 habitats. However, static interactions between habitats in the use of overlap areas did not differ (F_11,129 = 1.63, P = 0.09). Rabies antibody titers were highest in the managed pine habitat (28%) even prior to oral vaccine bait distributions in spring of 2004 and 2005. Biomarker data from radiocollared and additional raccoons captured after the bait distribution west of the Alabama River demonstrated a low efficacy of the vaccine reaching the small southern raccoons. The combination of the river as a partial barrier, the high percentage of pine forested habitat west of the river, and limited spatial movements of raccoons within these forested habitats appears to have reduced the likelihood of rabies establishing west of the river. Understanding different host-habitat- disease systems is important for successful management of diseases. Based on our results, we recommend that the oral vaccine program continue to use the Alabama River as a partial barrier and baiting be concentrated in the fragmented bottomland hardwood forests and around larger bodies of water where raccoon densities are highest. Success of baiting strategies designed to take advantage of northern raccoon dynamics and habitat use may not be applicable to southern populations.     

精制原代地鼠肾细胞狂犬病疫苗制备工艺的研究

通过ａＧ 株病毒在金黄地鼠肾细胞中培养,而制备的一种新型灭活狂犬病精制疫苗已获成功。该纯化方法包括醋酸锌沉淀和Ｓｅｐｈａｒｏｓｅ ４ＦＦ柱层析,所生产的疫苗质量完全达到新型狂犬病疫苗的要求。该工艺方法简单,成本低廉,是一种理想的狂犬病疫苗生产方法。     

Establishment and preliminary application of a rapid fluorescent focus inhibition test (RFFIT) for rabies virus

The World Health Organization (WHO) standard assay for determining levels of the rabies virus neutralization antibody (RVNA) is the rapid fluorescent focus inhibition test (RFFIT), which is used to evaluate the immunity effect after vaccination against rabies. For RFFIT, CVS-11 was used as the challenge virus, BSR cells as the adapted cells, and WHO rabies immunoglobulin (WHO STD) as the reference serum in this study. With reference to WHO and Pasteur RFFIT procedures, a micro-RFFIT procedure adapted to our laboratory was produced, and its specificity and reproducibility were tested. We tested levels of RVNA in human serum samples after immunization with different human rabies vaccines (domestic purified Vero cell rabies vaccine (PVRV) and imported purified chick embryo cell vaccine (PCECV)) using different regimens (Zagreb regimen and Essen regimen). We analyzed the levels of RVNA, and compared the immune efficacy of domestic PVRV and imported PCECV using different immunization regimens. The results showed that the immune efficacy of domestic PVRV using the Zagreb regimen was as good as that of the imported PCECV, but virus antibodies were generated more rapidly with the Zagreb regimen than with the Essen regimen. The RFFIT procedure established in our laboratory will enhance the comprehensive detection ability of institutions involved in rabies surveillance in China.     

The efficacy of a cultured rabies vaccine studied in guinea pigs preliminarily infected with a fixed rabies virus]

In experiments of curative vaccination, carried out with the use of an experimental model similar to the current practice of treatment with antirabies preparations, the advantages of using tissue-culture rabies vaccine with immunogenic potency equal to 1.3 international units (I. U.) were shown. In these experiments the vaccine was introduced into guinea pigs infected with fixed rabies virus, the course of vaccination consisting of 14 daily injections. No correlation between the induction of virus-neutralizing antibodies and the immunogenic potency of tissue-culture rabies vaccine was established: the use of the vaccine with immunogenic potency equal to 0,3 and 1,3 I.U. had no essential influence on the level of antibody formation in the animals.     

ECVAM-ICCVAM: prospects for future collaboration

The level and complexity of testing for hazard and risk assessment of marketed products and environmental agents has increased substantially over time, resulting in the use of greater numbers of both animals and humans for testing. Today, industry and regulatory bodies worldwide face increasing pressures to demonstrate responsible utilisation of laboratory animals, to limit their use, and to employ alternative non-animal tests. Institutions have also been established to identify, encourage development of, conduct research on, and validate new, improved, and surrogate test methods that will reduce and replace animal use. Two such organisations are ECVAM and the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM). As the evolutionary changes occurring in the field of toxicology result in an unprecedented increase in the introduction of alternative methodologies, these will strain the capacities of such alternative methods institutions. That realisation is causing a shift in thinking and creating an impetus to seek approaches by which to collaborate and develop more-efficient operational procedures for the validation and regulatory acceptance of alternative methods. Similarities in objectives, functions, scientific standards, and commitment to the principles of validation and animal welfare support the value of a cooperative arrangement between ECVAM and ICCVAM, to minimise duplication of effort, maximise productivity, and influence the international adoption of alternative tests. Opportunities for ECVAM-ICCVAM collaboration are discussed, which illustrate the feasibility and potential benefits of such a partnership.     

ECVAM's contributions to the implementation of the Three Rs in the production and quality control of biologicals

A summary is presented of the activities initiated, and the progress achieved, between April 1993 and December 2001 in implementing the Three Rs in one of the main priority areas of the European Centre for the Validation of Alternative Methods (ECVAM) - the production and quality control of biologicals. These have included organising eight key workshops, and financial contributions to, and sponsorship of, relevant international workshops, symposia and conferences. Noteworthy activities include financial support and/or participation in a number of prevalidation and validation studies. These involved alternative methods for the batch potency testing of: human tetanus vaccines; human and veterinary tetanus antisera and immunoglobulin; rabies vaccines; Leptospira hardjo vaccines; Clostridium perfringens vaccines; and erysipelas vaccines. They also involved a cell culture test for specific toxicity testing of diphtheria toxoid vaccines. In addition, ECVAM funded a study on the use of humane endpoints for vaccine quality control tests involving severe suffering, such as the potency testing of erysipelas, rabies and pertussis vaccines. ECVAM has also contributed financially to the compilation of manuals and expert reports, and to training in test methods. Following the report of an ECVAM Task Force, ECVAM financially supported the prevalidation of some in vitro methods for the potency testing of a recombinant hormone. A proposal is presented for promotion of regulatory acceptance, and suggestions are made for possible future activities.     

Effect of specific antibodies on active immunity development in those inoculated with an antirabies vaccine

The study of the effect of gamma globulin introduced in different doses (0.5 and 0.25 ml/mg) in combination with Fermi rabies vaccine (observations on humans were made) and with cerebral rabies vaccine inactivated by UV irradiation (in animal experiments) demonstrated that the injection of the higher doses of gamma globulin resulted in lower geometrical mean of antibody titers. Therefore, in combined administration of rabies vaccine and gamma globulin for postexposure rabies prevention it is advisable to reduce the dose of gamma globulin by one-half.     

Prevalence of tetracycline and rabies virus antibody in raccoons, skunks, and foxes following aerial distribution of V-RG baits to control raccoon rabies in Ontario, Canada

More than 3.6 million baits containing a recombinant vaccinia virus-rabies glycoprotein (V-RG) oral rabies vaccine were aerially or hand-distributed during 1999-2006 in an approximate 4,000-9,000 km(2) area of eastern Ontario, Canada, as part of a multitactic approach to control the raccoon variant of rabies. The efficacy of the program was assessed through the collection and testing of > 6,900 animals for bait acceptance and rabies virus-specific antibodies. Raccoon acceptance of rabies vaccine baits was significantly greater (71-83% ) in areas baited at a density of 150 baits/km(2) compared to areas baited at 75 baits/km(2) (26-58% ), and more raccoons consumed vaccine baits in areas baited with a flight line spacing of 0.75 km (45.3% [321/708]) than with a spacing of 1.5 km (33.8% [108/320]). In addition, greater numbers of raccoons consumed vaccine baits during a drop in September (52.7% [213/404]) as opposed to a June bait drop (34.6% [216/624]). Seropositivity rates for raccoons ranged between 7% and 28% in areas baited at 75/km(2) and 10% to 27% in areas baited at 150/km(2) with statistical differences varying among years and treatments. The last case of raccoon-variant rabies reported in Ontario was in September 2005. The control of raccoon rabies in Ontario has resulted in an estimated $6M to $10 M Cdn annual savings in rabies-associated costs.