Humoral Immune Response of Thai Dogs after Oral Vaccination against Rabies with the SPBN GASGAS Vaccine Strain

Abstract

Applied research is crucial in pushing the boundaries and finding a solution to the age-old problem of dog-mediated rabies. Although oral vaccination of dogs is considered to have great potential in mass dog vaccination campaigns and could have far-reaching benefits, it is perhaps the most ignored of all available tools in efforts to eliminate dog-mediated rabies, not least because of limited data on immunogenicity, efficacy, and safety of potential oral rabies vaccine candidates. In this study, the long-term immunogenicity in local Thai dogs after oral administration of the highly attenuated 3rd generation rabies virus vaccine strain SPBN GASGAS was assessed. The oral rabies vaccine was administered to dogs by either direct oral administration (n = 10) or by offering a vaccine loaded intestine bait (n = 15). The humoral immune response was then compared to three groups of dogs; a group that received a parenteral delivered inactivated rabies vaccine (n = 10), a group offered a placebo intestine bait (n = 7), and a control group (n = 4) for an observation period of 365 days. There was no significant difference in the immune response of dogs that received oral and parenteral vaccine in terms of magnitude, kinetics, and persistence of both rabies virus (RABV) neutralizing (RFFIT) and binding (ELISA) antibodies. Although the single parenteral injection of an inactivated rabies vaccine mounted a slightly higher humoral immune response than the orally delivered live vaccine, RABV specific antibodies of both types were still detectable after one year in most animals for all treatment groups and resulted in no difference in seropositivity. Characterization of rabies specific antibodies revealed two main classes of antibodies involved in the immune response of dogs vaccinated. While IgM antibodies were the first to appear, the succeeding IgG response was mainly IgG2 dominated independent of the vaccine type used. The results support the view that SPBN GASGAS induces a sustained detectable immune response in local dogs both after direct oral administration and via bait application.

Keywords: SPBN GASGAS; dogs; immunoglobuline isotypes; neutralizing and binding antibodies; oral vaccination.

Figure 1

Study design and animal husbandry. (a) Outline of the in vivo experiments with an observation period of 365 days. (b) Cage allocation of individual animals according to treatment groups: (Group A—blue [bait], Group B—purple [doa], Group C—orange [sc], Group D—red [placebo] and Group E—green [control]). (c) Locally produced boiled pig intestine bait (upper picture) and vaccine sachets inserted in a previously cleaned and boiled section of the cow intestine (lower picture). (d) Standard animal cages at the dog shelter in Taptan, Uthai-Thani. Cages had a size of 90 × 350 × 190 cm (length × width × height) accessible through an open fenced door. The floor and lower part of the walls (100 cm) was made from concrete, and the upper part of the walls consisted of fencing.

Figure 2

Antibody response in local Thai dogs after oral rabies vaccination (Group A and B), parenteral vaccination (Group C) using SPBN GASGAS and a commercial inactivated vaccine, respectively, as well as after placebo baiting (Group D) and left untreated (Group E). Serology results of all treatment groups are presented as mean percent inhibition and geometric mean titer with standard deviation as measured by ELISA (a) and Rapid Fluorescent Focus Inhibition Test (RFFIT) (b), respectively.

Figure 3

Kinetics of RABV specific immunoglobulin classes (IgM, IgG, IgA) and IgG isotypes (IgG1, IgG2) in the serum of naïve dogs (Group E) compared to orally (Group A) and parenterally vaccinated dogs (Group C) at different time points (days) pv. Each dot represents one individual animal with the median (solid line) depicted for each time point and each group. The vertical dashed lines represent the time point of vaccination. Statistical comparisons of the means for IgM (14 dpv), IgG (28 dpv), IgG₂ (28 dpv) between the different groups are indicated (Fisher’s t-test, p < 0.05, *** = extremely significant p: 0.0001 to 0.001; NS: not significant).

Figure 4

Comparison of correlation between optical density (OD) values of RABV specific immunoglobulins (IgG isotypes) and binding as well as VNAs after parenteral vaccination with an oral rabies vaccine (SPBN GASGAS) between orally (Group A and B) and parenterally (Group C) vaccinated animals. Correlation of RABV-specific IgG (a,b), and IgG2 (c,d) with rVBA as measured by ELISA. Correlation of RABV-specific IgG (e,f), and IgG2 (g,h) with rVNAs as measured by RFFIT. Each dot represents the results of one individual animal at different time points pv. Linear regression lines (solid lines) and the p-values are indicated.

Figure 5

Correlation between ELISA (y-axis) and RFFIT (x-axis) results. The dotted lines represent the threshold of positivity in ELISA (horizontal; 40% PI) and RFFIT (vertical, 0.5 IU/mL). Sectors from both assays that are considered negative and positive are highlighted in light green and light red, respectively.