Evaluation of the safety and immunogenicity of the ERAGS-GFP oral rabies vaccine in dogs

Abstract

Rabies is a fatal zoonotic disease primarily transmitted through dog bites, making oral rabies vaccines critical for disease control. This study evaluated the immunogenicity of the ERAGS-GFP oral rabies vaccine strain in dogs. To optimize viral production, we examined cell density, multiplicity of infection (MOI), and freeze-thaw cycles. Safety was assessed via clinical monitoring, body temperature, and weight changes. Immunogenicity was evaluated using rabies virus neutralizing antibody (VNA) titers. Vero cells at MOI 2 with 3 freeze-thaw cycles yielded the highest viral titers. Vaccinated dogs showed no clinical symptoms and developed sustained protective VNA titers, demonstrating ERAGS-GFP’s efficacy in rabies control.

Rabies remains one of the deadliest zoonotic diseases, posing a severe threat to global human and animal health. It is primarily transmitted through the bites of rabid dogs, which account for the majority of human cases. Recognizing the urgent need for rabies elimination, international organizations including the World Health Organization have set a goal to eradicate dog-mediated rabies by 2030 [1]. Countries worldwide have implemented national rabies eradication programs involving mass vaccination, early reporting, strengthened surveillance, and quarantine measures. Despite these efforts, rabies continues to cause approximately 59,000 deaths annually [2]. Achieving successful rabies eradication requires vaccinating over 70% of the canine population. However, a key challenge is capturing free-roaming dogs, especially in endemic regions [3]. Effective management of stray dogs is crucial for rabies control, and oral rabies vaccination (ORV) offers a promising solution by enabling immunization of difficult-to-capture populations [4].

ORV is a widely utilized preventive measure for rabies control, particularly in wild and free-roaming dogs. Upon ingestion, the vaccine virus infects the mucosal membranes and tonsils, inducing an immune response [5]. Early ORV strains (ERA, SAD Bern, and SAD B19) were attenuated through multiple passages, but exhibited residual pathogenicity, causing vaccine-associated rabies cases [6,7,8,9]. The second-generation strain, SAG-2, was developed through monoclonal antibody selection, demonstrating effective immunity in dogs and foxes [10]. Third-generation ORV strains, RABORAL V-RG^® (Boehringer Ingelheim, Ridgefield, CT, USA) and ONRAB^® (Artemis Technologies, Guelph, ON, Canada), utilize recombinant viral vectors and have successfully prevented rabies in wild populations [11,12]. The latest strain, SPBN GASGAS, was designed using reverse transcription for precise genetic modifications to enhance safety, preventing rabies in raccoons and foxes [13].

In the Republic of Korea (ROK), dog-mediated rabies has been eradicated since 1980 due to extensive vaccination programs. However, since 1993, rabid raccoon dogs have caused 179 cases in cattle, 177 in dogs, 76 in raccoons, 4 in cats, and one in deer [14]. To mitigate this risk, RABORAL V-RG® bait vaccines have been deployed in rabies-risk regions since 2002, maintaining a rabies-free status since 2014 [14]. Enhancing vaccination coverage in free-roaming dogs is vital to sustaining this status. ORVs have been proposed as an alternative to parenteral vaccination, following successful implementation in wild animal populations [8]. In a previous study, we developed the ERAGS-GFP strain through reverse transcription, introducing fluorescent protein expression while modifying pathogenic amino acids [15]. The strain demonstrated non-pathogenicity in mice following intracranial injection. This study aims to optimize viral production conditions for ERAGS-GFP and evaluate its safety and immunogenicity in dogs.

The ERAGS-GFP strain was deposited in the Korean Veterinary Culture Collection (KVCC-VR1900060) and propagated in Vero cells (ATCC, Manassas, VA, USA) for use as an ORV strain. The CVS-11 strain was propagated in BHK-21 cells (ATCC) for the rabies virus neutralizing antibody (VNA) test. Vero, Neuro-2a, and BHK-21 cells were utilized to determine optimal viral titers and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5%–10% fetal bovine serum. BHK-21, Neuro-2a, and Vero cells were seeded at a density of 200,000 cells/mL into 25 cm² flasks and incubated for 24 hours. The ERAGS-GFP strain was inoculated at MOIs of 0.1, 1, 2, 4, and 10, and incubated for 5 days. To assess virus stability, infected Vero cells underwent 1, 2, or 3 freeze-thaw cycles before supernatant collection. Virus titers were measured using the Reed and Muench method. Infected cells were identified by fluorescence microscopy, and viral titers were determined at day 5 post-infection. The titer of ERAGS-GFP administered orally or subcutaneously was 10^8.0 TCID₅₀/mL (Table 1). All procedures were approved by the Institutional Animal Care and Use Committees (IACUCs approval number: 2024-279). Fourteen dogs were divided into 3 experimental groups. Six dogs received oral ERAGS-GFP three times at 2-week intervals, 6 received a single subcutaneous injection, and 2 remained untreated as controls. Body temperature and weight were recorded at baseline and at 4, 8, and 10 weeks post-vaccination. Clinical signs were monitored daily for 10 weeks. Blood samples were collected at 0, 2, 4, 6, and 10 weeks post-administration to measure rabies antibody titers. Rabies VNA titers were determined using a fluorescent antibody virus neutralization assay, employing a reference serum with a titer of 0.5 IU/mL. Rabies antibodies were also quantified using a blocking enzyme-linked immunosorbent assay (ELISA) kit (BioPro Rabies ELISA Ab Kit; BioPro, Praha, Czech Republic). Serum samples with percentage blocking (PB) <40% were considered negative, PB ≥40% were positive, and PB ≥70% indicated protective antibody levels. All values were expressed as the mean ± standard deviation. Statistical analysis was conducted using GraphPad Prism Software version 5.0 (GraphPad, San Diego, CA, USA). One-way analysis of variance followed by Dunnett’s multiple comparison test was performed to determine significance, with p<0.001, p<0.01, and p<0.05 considered statistically significant.

Table 1. Specific primer sets for rabies virus diagnosis.

Designation	Oligonucleotide	Expected size (bp)	Target gene	Remarks
JW12	ATGTAACACCY*CTACAATG	606	N
JW6UNI	CAR*TTVGCRCACATYTTRTG

^*Y, C or T; R, A or G.

Stable, high-titer production of the ERAGS-GFP strain is essential for supplying sufficient vaccine antigen. To identify optimal cell lines and MOI for ERAGS-GFP production, three cell types were infected with 5 different MOIs, and virus titers were measured. BHK-21, Neuro-2a, and Vero cells inoculated with the 5 MOIs yielded average viral titers of 10^8.0, 10^7.4, and 10^8.1 TCID₅₀/mL, respectively (Fig. 1A). Consequently, Vero cells inoculated with an MOI of 2 were selected for ERAGS-GFP production. To release cell-associated rabies virus, the harvested virus was subjected to freeze-thaw cycles. The titers of the ERAGS-GFP strain after the freeze-thaw cycles were 10^7.8,7.8,8.0, and 10^8.3 TCID₅₀/mL, respectively (Fig. 1B). Thus, three freeze-thaw cycles were determined to be optimal. The safety of the ERAGS-GFP strain administered orally or subcutaneously was monitored in dogs for 10 weeks. No clinical signs of rabies were observed in any dogs, including controls (Table 2). Body temperatures of orally and subcutaneously vaccinated dogs were measured for 7 days, with average values remaining below 40°C (Fig. 2A). The average body weight of vaccinated dogs increased up to 8 weeks and then stabilized until 10 weeks, whereas the control group’s weight continued to rise (Fig. 2B). Dogs administered the ERAGS-GFP strain orally developed mean rabies VNA titers of 0.58, 0.93, 1.71, and 1.87 IU/mL at 2, 4, 6, and 10 weeks post-administration, respectively. Those receiving a single subcutaneous injection showed mean rabies VNA titers of 0.17, 3.40, 5.77, and 5.39 IU/mL at the same intervals. Control dogs had a mean titer of 0.06 IU/mL at 10 weeks, indicating no rabies antibodies (Fig. 3A). The same serum samples used in the fluorescent antibody virus neutralization test were assessed in a blocking ELISA. Orally vaccinated dogs had mean PB of 61, 80, 83, and 82 at 2, 4, 6, and 10 weeks, respectively. Subcutaneously vaccinated dogs showed PBs of 60, 88, 92, and 93 at the same intervals. Control dogs had PBs ranging from 0 to 13 over 10 weeks (Fig. 3B).

Fig. 1. Comparative proliferation of ERAGS-GFP in cell lines and MOI condition. (A) Proliferation ability of the ERAGS-GFP strain in 3 cell types at different multiplicities of infection. Among the 3 cell lines, Vero cells inoculated at 2 MOI showed the highest viral titer of the ERAGS-GFP strain. (B) Viral titers of the ERAGS-GFP strain after different numbers of freeze-thaw cycles. The strain subjected to 3 freeze-thaw cycles achieved the highest viral titer.

MOI, multiplicity of infection.

Table 2. Clinical signs in dogs administered the ERAGS-GFP strain over 10 weeks.

Animal species	No. of animals	Route/Titer*	Clinical signs for 10 weeks
			Neurologic	Salivation	Behavior	Diarrhea	Anorexia
Dogs	6	Oral/108.0	0/6	0/6	0/6	0/6	0/6
	6	SC/108.0	0/6	0/6	0/6	0/6	0/6
	2	No treatment	0/2	0/2	0/2	0/2	0/2

SC, subcutaneous injection.

^*10^8.0 TCID₅₀/mL.

Fig. 2. Changes in the average body temperature (A) and body weight (B) of dogs administered the ERAGS-GFP strain.

SC, subcutaneous injection.

Fig. 3. Immunogenicity assessment of ERAGS-GFP strain using VNA test and ELISA. (A) Mean rabies VNA titers in dogs administered the ERAGS-GFP strain. At 2 weeks post-oral administration, the average VNA titer exceeded the protective level of 0.5 IU/mL. (B) PB on ELISA. PB 40 (dotted line) and 70 (solid line) indicate the thresholds for rabies antibody positivity and a protective antibody level, respectively. Statistical significance among groups was assessed by one-way analysis of variance.

VNA, virus neutralizing antibody; PB, percentage blocking; ELISA, enzyme-linked immunosorbent assay; SC, subcutaneous injection.

^*p<0.05, ^**p<0.01, ^***p<0.001, ^****p<0.0001.

ORV is a practical strategy to enhance vaccination coverage among aggressive or difficult-to-capture dogs [16]. It is cost-effective and applicable to various animal species. However, ORV vaccine strains can be shed from a dog’s mouth post-administration, increasing potential exposure to immune tissues. The effectiveness of ORV largely depends on viral titers, with current bait vaccines such as SAG-2, V-RG, AdRG1.3, and SPBN GASGAS containing titers above 10^8.0 TCID₅₀ per bait [3,11,12,13]. In this study, ERAGS-GFP inoculated in BHK-21 and Vero cells produced high viral titers exceeding 10^8.0 TCID₅₀/mL, making them suitable for large-scale production. Among these cell lines, Vero cells demonstrated optimal productivity and stability at an MOI of 2, preventing early saturation effects seen at MOI 10. Although viral titers were comparable at MOIs of 2 and 10, slightly lower yield at 10 MOI suggests early infection saturation, limiting subsequent rounds of viral replication. The safety of ORV strains has been extensively demonstrated in various species, including dogs, foxes, and raccoon dogs. Regulatory authorities require vaccine manufacturers to provide comprehensive safety data before approval. Previous studies have confirmed the safety of SAG-2 in foxes and raccoon dogs, and AdRG1.3 was found safe in multiple species at high oral doses [17,18]. In this study, dogs administered ERAGS-GFP either orally or subcutaneously maintained body temperatures below 40°C for 7 days, with no clinical rabies symptoms observed over 10 weeks. Additionally, ERAGS-GFP was commercialized as a bait vaccine for raccoon dogs in the ROK in 2014, further supporting its safety profile.

Rabies VNA are crucial for evaluating immune responses post-vaccination. Previous research on SAG-2 and SPBN GASGAS showed sustained VNA levels in vaccinated dogs [19,20]. In this study, ERAGS-GFP vaccinated dogs exhibited VNA titers increasing from 0.58 IU/mL at 2 weeks to 1.87 IU/mL at 10 weeks. Blocking ELISA confirmed the presence of rabies antibodies, indicating successful immune induction. However, further studies are required to assess long-term immunogenicity and optimize bait vaccine formulations for dogs.

In conclusion, the ERAGS-GFP strain demonstrated high-titer production in Vero cells at MOI 2, ensuring stability and effective viral replication. It was found to be safe for dogs over a 10-week period and successfully induced rabies-specific immunity. These results highlight its potential as an effective ORV candidate for canine rabies control, though additional research is needed to refine vaccination strategies.