To test the immunogenicity and efficacy of a new oral rabies virus vaccine strain SPBN GASGAS in wildlife target species, one group of foxes and two groups of raccoon dogs were offered a bait containing 1.7 ml of the vaccine (10 FFU/ml; 10 FFU/dose) and subsequently challenged approximately 180 days later with a fox rabies virus isolate. One group of raccoon dogs (n=30) received the same challenge dose (10 MICLD/ml) as the red foxes (n=29). The other group with raccoon dogs (n=28) together with 8 animals that received the vaccine dose by direct instillation into the oral cavity (DIOC) were infected with a 40-fold higher dose of the challenge virus (10 MICLD/ml). All but one of the 29 vaccinated foxes survived the challenge infection; meanwhile all 12 control foxes succumbed to rabies. Twenty-eight of 30 vaccinated raccoon dogs challenged with the same dose survived the infection, however only six of 12 control animals succumbed. When the higher challenge dose was administered, all 12 control animals died from rabies and all 36 vaccinated animals (28 baited plus 8 DIOC) survived. Blood samples were collected at different time points post vaccination and examined by both RFFIT and ELISA. The kinetics of the measured immune response was similar for both species, although in RFFIT slightly higher values were observed in foxes than in raccoon dogs. However, the immune response as measured in ELISA was identical for both species. The oral rabies virus vaccine SPBN GASGAS meets the efficacy requirements for live rabies virus vaccines as laid down by the European Pharmacopoeia.

In many arthropods, such as bushcrickets, males donate protein-rich nuptial gifts-so-called spermatophores-to females, which females ingest while the sperm enter the female's reproductive tract. Previously, it was shown that females route spermatophore nutrients over the course of hours and days to egg production or body synthesis. We investigated whether female bushcrickets fuel their metabolism with spermatophores immediately after consumption. We fed two male groups diets that were either enriched or depleted in 13 C, and then tracked the isotopic changes in exhaled breath in female bushcrickets after spermatophore consumption. Within 3 hours, the stable carbon isotope ratio (d 13 C) of female breath converged on the ratio of the male donor of the nuptial gift. This supports the idea that females quickly routed nutrients to metabolism, receiving immediate benefits from spermatophore feeding.

: To evaluate the long-term immunogenicity of the live-attenuated, oral rabies vaccine SPBN GASGAS in a full good clinical practice (GCP) compliant study, forty-six (46) healthy, seronegative red foxes (Vulpes vulpes) were allocated to two treatment groups: group 1 (n = 31) received a vaccine bait containing 1.7 ml of the vaccine of minimum potency (106.6 FFU/mL) and group 2 (n = 15) received a placebo-bait. In total, 29 animals of group 1 and 14 animals of group 2 were challenged at 12 months post-vaccination with a fox rabies virus isolate (103.0 MICLD50/mL). While 90% of the animals offered a vaccine bait resisted the challenge, only one animal (7%) of the controls survived. All animals that had seroconverted following vaccination survived the challenge infection at 12 months post-vaccination. Rabies specific antibodies could be detected as early as 14 days post-vaccination. Based on the kinetics of the antibody response to SPBN GASGAS as measured in ELISA and RFFIT, the animals maintained stable antibody titres during the 12-month pre-challenge observation period at a high level. The results indicate that successful vaccination using the oral route with this new rabies virus vaccine strain confers long-term duration of immunity beyond one year, meeting the same requirements as for licensure as laid down by the European Pharmacopoeia.

ABSTRACT:The small Indian mongoose (Herpestes auropunctatus), a rabies reservoir species on several Islands in the Caribbean, was successfully immunized against rabies for the first time by offering animals a vaccine bait specifically designed for this small carnivore. The bait contained on average 0.6 mL of the genetically modified replication-competent rabies virus construct SPBN GASGAS (10 8.5 focus-forming units/mL). Three of four mongooses offered a bait developed an immune response above 0.5 IU/mL, but the response was less pronounced than in two animals offered the vaccine by direct oral instillation.

Oral rabies vaccination (ORV) is highly effective in foxes and raccoon dogs, whereas for unknown reasons the efficacy of ORV in other reservoir species is less pronounced. To investigate possible variations in species-specific cell tropism and local replication of vaccine virus, different reservoir species including foxes, raccoon dogs, raccoons, mongooses, dogs and skunks were orally immunised with a highly attenuated, high-titred GFP-expressing rabies virus (RABV). Immunofluorescence and RT-qPCR screenings revealed clear differences among species suggesting host specific limitations to ORV. While for responsive species the palatine tonsils (tonsilla palatina) were identified as a main site of virus replication, less virus dissemination was observed in the tonsils of rather refractory species. While our comparison of vaccine virus tropism emphasizes the important role that the tonsilla palatina plays in eliciting an immune response to ORV, our data also indicate that other lymphoid tissues may have a more important role than originally anticipated. Overall, these data support a model in which the susceptibility to oral live RABV vaccine infection of lymphatic tissue is a major determinant in vaccination efficacy. The present results may help to direct future research for improving vaccine uptake and efficacy of oral rabies vaccines under field conditions.

A safety requirement for live vaccines is investigating possible shedding in recipients since the presence of replication competent vaccine in secretions could result in direct and indirect horizontal transmission. This is especially relevant for oral rabies vaccine baits that are deliberately distributed into the environment. In the current study, survival of an oral rabies virus vaccine, SPBN GASGAS, was examined in excretions from different target and non-target species; red fox, raccoon dog, small Indian mongoose, raccoon, striped skunk, domestic dog, domestic cat and domestic pig. Saliva - and (pooled) fecal samples collected at different time points after oral administration of the vaccine strain were examined for the presence of viral RNA (rt-PCR). All PCR-positive and a subset of PCR-negative samples were subsequently investigated for the presence of infectious virus by isolation in cell culture (RTCIT). Up to 7 days post vaccine administration viral RNA could be detected in 50 of 758 fecal samples but no infectious virus was detected in any of the examined PCR-positive fecal samples. In contrast, RNA-fragments were detected in 248 of 1053 saliva swabs for an extended period (up to 10 days) after vaccine administration, but viable virus was only present during the first hours post vaccine administration in 38 samples. No infectious vaccine virus was isolated in saliva swabs taken 24 h or more after vaccine administration. Hence, no active shedding of the vaccine virus SPBN GASGAS after oral administration occurred and the virus isolated during the initial hours was material originally administered and not a result of virus replication within the host. Thus, potential horizontal transmission of this vaccine virus is limited to a short period directly after vaccine bait uptake. It can be concluded that the environmental risks associated with shedding after distributing vaccine baits containing SPBN GASGAS are negligible.

BackgroundOral vaccination of the small Indian mongoose against rabies has been suggested as a potential tool to eliminate mongoose-mediated rabies on several Caribbean islands. A recently developed oral rabies virus vaccine strain, SPBN GASGAS, has already been shown to be efficacious in this reservoir species. Since, all available oral rabies vaccines are based on replication-competent viruses and vaccine baits are distributed unsupervised in the environment, enhanced safety standards for such vaccine types are required.ResultsThe results of safety studies, including overdose, repeated doses, dissemination and different routes of administration, in the target species are presented. It was shown that the construct was apathogenic, irrespective of dose and route of administration. Even when it was inoculated directly in the brain, it did not induce rabies infection. Furthermore, the vaccine strain did not spread within the target species after direct oral instillation beyond the site of entry.ConclusionThe vaccine strain SPBN GASGAS meets the safety requirements for live rabies virus vaccines in this target species, the small Indian mongoose.

In order to obtain Marketing Authorization for an oral rabies vaccine in the European Union, not only safety studies in the target species, red fox and raccoon dog, are required. Since baits are distributed unsupervised in the environment, specific safety studies in selected non-target species are compulsory. Furthermore, oral rabies vaccines are based on live, replication-competent viruses and thus distinct safety studies in the target species for such type of vaccines are also mandatory. Here, the results of these safety studies in target and selected non-target species for a 3rd generation oral rabies virus vaccine construct, SPBN GASGAS (Rabitec), are presented. The studies included the following species; red fox, raccoon dog, domestic dog, domestic cat, domestic pig, wild rodents. The following safety topics were investigated; overdose, repeated dose, dissemination, shedding, horizontal and vertical transmission. It was shown that SPBN GASGAS did not cause disease or any other adverse reaction in vaccinated animals and naïve contact animals. The vaccine did not disseminate within the host beyond the site of entry. No horizontal transmission was observed in wild rodents. In the target species, there was evidence that in a few cases horizontal transmission of vaccine virus could have occurred under these experimental conditions; most likely immediately after vaccine administration. The vaccine construct SPBN GASGAS meets therefore the latest revised minimal safety requirements as laid down in the European Pharmacopoeia.