Rabies Prophylactic and Treatment Options: An In Vitro Study of siRNA- and Aptamer-Based Therapeutics

If the goal of eliminating dog-mediated human rabies by 2030 is to be achieved, effective mass dog vaccination needs to be complemented by effective prophylaxis for individuals exposed to rabies. Aptamers and short-interfering RNAs (siRNAs) have been successful in therapeutics, but few studies have investigated their potential as rabies therapeutics. In this study, siRNAs and aptamers—using a novel selection method—were developed and tested against rabies virus (RABV) in a post-infection (p.i.) scenario. Multiple means of delivery were tested for siRNAs, including the use of Lipofectamine and conjugation with the developed aptamers. One siRNA (N53) resulted in an 80.13% reduction in viral RNA, while aptamer UPRET 2.03 demonstrated a 61.3% reduction when used alone at 2 h p.i. At 24 h p.i., chimera UPRET 2.03-N8 (aptamer-siRNA) resulted in a 36.5% inhibition of viral replication. To our knowledge, this is the first study using siRNAs or aptamers that (1) demonstrated significant inhibition of RABV using an aptamer, (2) tested Lipofectamine RNAi-Max as a means for delivery, and (3) produced significant RABV inhibition at 24 h p.i. This study serves as a proof-of-concept to potentially use aptamers and siRNAs as rabies immunoglobulin (RIG) replacements or therapeutic options for RABV and provides strong evidence towards their further investigation.     

Human benefits of animal interventions for zoonosis control

Although industrialized countries have been able to contain recent outbreaks of zoonotic diseases, many resource-limited and transitioning countries have not been able to react adequately. The key for controlling zoonoses such as rabies, echinococcosis, and brucellosis is to focus on the animal reservoir. In this respect, ministries of health question whether the public health sector really benefits from interventions for livestock. Cross-sectoral assessments of interventions such as mass vaccination for brucellosis in Mongolia or vaccination of dogs for rabies in Chad consider human and animal health sectors from a societal economic perspective. Combining the total societal benefits, the intervention in the animal sector saves money and provides the economic argument, which opens new approaches for the control of zoonoses in resource-limited countries through contributions from multiple sectors.     

Knowledge, attitudes, and practices survey of rabies in a community in Sri Lanka

Objectives The primary objective of this study was to determine the level of knowledge, attitudes and practices (KAP) of rabies management and control of a sample population. The secondary objective was to compare the KAP with respect to rabies management and control between urban and rural areas and between pet and non-pet owners. Methods This cross-sectional study was carried out by conducting face-to-face interviews using structured questionnaires among 1570 respondents from selected households in the Kandy District, Sri Landa. Results Approximately 58% of the sample population was pet owners. Among all the respondents, there was a high level of awareness (90%) that dogs are the most common rabies reservoir, that the disease is fatal (79%), and that rabies can be prevented by vaccination (88%). Most of the subjects (96%) would seek treatment from a doctor or a hospital after being bitten by a dog. Although 76% of the respondents said that their pet dogs were vaccinated, only one-half were able to present a vaccination certificate upon request. The subjects from the urban areas would submit the head of an animal for rabies evaluation (69%) compared with those from the rural areas (57%). Pet owners (93%) are more aware that dog rabies vaccines are available from authorized offices than non-pet owners (87%). Conclusions The level of awareness of rabies and the level of receptiveness to rabies control measures are high. There is a difference in the attitudes and pet care practices relevant to rabies control between urban and rural areas. Pet owners tend to be more cooperative to rabies control activities. The attitudes and practices of the respondents may reflect the inaccessibility of facilities and the lack of services that would enable community participation in rabies control.     

A novel in vitro ELISA for estimation of glycoprotein content in human rabies vaccines

In vitro methods for quantification of immunodominant glycoprotein in the rabies vaccine formulations serve as good alternative to the cumbersome and variable mice potency assay as a batch release test for the vaccine. The present study presents the development of a sandwich ELISA with optimal concentrations of a high affinity recombinant diabody (D06) and a specific monoclonal antibody (M5B4) against rabies glycoprotein for its quantification in the vaccine formulations. The glycoprotein estimate correlated linearly (r² = 0.8) to the in vivo potency estimate for the vaccine formulations. This ELISA promises a good forecast of the mice potency values and thereby can serve as a simple, yet effective batch release test for the rabies vaccines replacing the in vivo assay.     

Ikoma lyssavirus, highly divergent novel lyssavirus in an African civet

Evidence in support of a novel lyssavirus was obtained from brain samples of an African civet in Tanzania. Results of phylogenetic analysis of nucleoprotein gene sequences from representative Lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species, designated Ikoma lyssavirus.     

成大速达与瑞必补尔人用狂犬病疫苗四针免疫法安全性比较研究

目的为了全面了解国产与进口人用狂犬病疫苗四针免疫法的安全性。方法对济南铁路疾控中心接种门诊狂犬病疫苗接种对象的疫苗反应进行分析,比较研究国产与进口人用狂犬病疫苗四针免疫法的疫苗安全性。结果国产苗102人发生了39例接种反应,反应率38．24％;进口苗93人发生了27例接种反应,反应率29．03％;差别没有统计学意义。这些反应以一般反应为主,症状轻微,不治而愈;说明两组的疫苗安全性大体一致。但是,疼痛、红肿、年龄〈20岁组的疫苗反应率,国产苗高于进口苗。在国产疫苗组,年龄〈20岁组的疫苗反应率高于其它年龄组,4例体温超过38．6℃的均为10岁以内的儿童,重反应率为3．92％（4／102）,需要对症治疗;并且,这个年龄组的疫苗反应持续时间较长。结论尽管国产狂犬病疫苗四针法的安全性较好,但对于较小年龄的接种对象,应尽量使用进口苗,以避免不良反应的发生,确保疫苗的使用安全。     

Serological methods used for rabies post vaccination surveys: An analysis

The assessment of fox immunity following oral rabies vaccination (ORV) is commonly applied to assess the efficacy of an ORV campaign in the field. Several ELISA kits have been developed and validated for their use for rabies serology in wildlife as an alternative to neutralizing techniques (NT), such as the fluorescent antibody virus neutralization test (FAVN) and the rapid fluorescent foci inhibition test (RFFIT). At a European level, NT and ELISA tests are used interchangeably and on different types of samples collected for vaccination follow-up. This has resulted in a difficulty in comparing the results generated with different diagnostic tools. We have evaluated (a) the effect of two different matrices commonly used for serology in red foxes on the results of the FAVN and (b) the performance of two commercially available ELISAs in comparison with the FAVN, as a gold standard, using a panel of over 700 field fox samples. Moderate agreement was observed when comparing results from different matrices. We found a very low level of agreement and low values of relative sensitivity and specificity of the ELISAs tested in comparison with the FAVN. Our findings confirm, using a vast collection of field samples obtained during post-vaccination surveillance campaigns in Italy, the need for improved reliability of certain serological tests.     

One clinic visit for pre-exposure rabies vaccination (a preliminary one year study)

We performed an abbreviated prospective study of rabies pre-exposure (PREP) vaccination in 109 volunteers. Group 1, the control group, received the conventional 3 intradermal injections on days 0, 7 and 21. Group 2 received one rabies vaccine injection (0.1 ml intradermally) at 2 sites on a single day. Group 3 was given one full ampule intramuscularly. One year later, all 3 groups received booster injections (0.1 ml at 4 sites) intradermally at one time or 2 injections intramuscularly on days 0 and 3. All subjects achieved a vigorous anamnestic antibody response 7 days after the boosters. These data suggest that one time immunization of one full dose intramuscularly or 2 site injections of 0.1 intradermally on a single day are adequate to prime immune memory and obtain an accelerated immune response one year later.     

Induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein

The introduction of exogenous genes into plants permits the development of a new generation of biological products, i.e., edible vaccines. Cereals, especially maize, have been the systems of choice for the expression of antigenic proteins because the proteins can be expressed at high levels in the kernel and stored for prolonged periods without excessive deterioration. The utilization of plant-derived antigens for oral delivery provides an alternative strategy for the control of pathogens in animals compared to the current vaccine administration methods, such as injection. However, there is some doubt about the efficacy of these types of vaccines in polygastric animals due to the features of their digestive system. Here, we report the efficacy of an edible vaccine against rabies evaluated in sheep. Kernels containing different doses of G protein (0.5, 1, 1.5 and 2mg) were given in a single dose by the oral route. Cumulative survival was better in groups that received 2mg of G protein and for the positive control (inactivated rabies vaccine); this observation was supported by the presence of neutralizing antibodies. Animals in the control group died after challenge. The degree of protection achieved for 2mg of G protein was comparable to that conferred by a commercial vaccine. In conclusion, this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model.