Inactivated Rabies Virus-Vectored Immunocontraceptive Vaccine in a Thermo-Responsive Hydrogel Induces High and Persistent Antibodies against Rabies, but Insufficient Antibodies against Gonadotropin-Releasing Hormone for Contraception

Abstract

Rabies is preventable through vaccination, but the need to mount annual canine vaccination campaigns presents major challenges in rabies control and prevention. The development of a rabies vaccine that ensures lifelong immunity and animal population management in one dose could be extremely advantageous. A nonsurgical alternative to spay/neuter is a high priority for animal welfare, but irreversible infertility in one dose has not been achieved. Towards this goal, we developed a rabies virus-vectored immunocontraceptive vaccine ERA-2GnRH, which protected against rabies virus challenge and induced >80% infertility in mice after three doses in a live, liquid-vaccine formulation (Wu et al., 2014). To improve safety and use, we formulated an inactivated vaccine in a thermo-responsive chitosan hydrogel for one-dose delivery and studied the immune responses in mice. The hydrogel did not cause any injection site reactions, and the killed ERA-2GnRH vaccine induced high and persistent rabies virus neutralizing antibodies (rVNA) in mice. The rVNA in the hydrogel group reached an average of 327.40 IU/mL, more than 200 times higher than the liquid vaccine alone. The Gonadotropin-releasing hormone (GnRH) antibodies were also present and lasted longer in the hydrogel group, but did not prevent fertility in mice, reflecting a possible threshold level of GnRH antibodies for contraception. In conclusion, the hydrogel facilitated a high and long-lasting immunity, and ERA-2GnRH is a promising dual vaccine candidate. Future studies will focus on rabies protection in target species and improving the anti-GnRH response.

Keywords: ERA-2GnRH; chitosan; gonadotropin-releasing hormone (GnRH); immunocontraceptive vaccine; nonsurgical sterilization; rabies virus; thermo-responsive hydrogel.

Figure 1

Gelling study of the ERA-2GnRH hydrogel vaccine. (a) Tube 1 is the ERA-2GnRH hydrogel at room temperature; the gel is liquid and settles down to the bottom. Tube 2 is the hydrogel vaccine incubated at 37 °C for 10 min. (b) Tube 1 is the hydrogel vaccine with a blue dye (0.01% Ivan’s blue) incubated at 37 °C for 10 min. Tube 2 is the room temperature product, a liquid gel.

Figure 2

Dynamics of rabies virus-neutralizing antibody titers after immunization in mice. (a) Geometric mean titer with standard deviation and (b) non-linear curve fit for each group at each time point are shown on a log-scale. Mice in Group A (n = 10) received no vaccine. Mice in Group B (n = 20) received inactivated ERA-2GnRH (Vaccine only). Mice in Group C (n = 20) received an equivalent dose of inactivated ERA-2GnRH in a chitosan hydrogel (Hydro). The end of the study (E) was approximately 70 days post-vaccination but varied for each group based on fertility.

Figure 2

Dynamics of rabies virus-neutralizing antibody titers after immunization in mice. (a) Geometric mean titer with standard deviation and (b) non-linear curve fit for each group at each time point are shown on a log-scale. Mice in Group A (n = 10) received no vaccine. Mice in Group B (n = 20) received inactivated ERA-2GnRH (Vaccine only). Mice in Group C (n = 20) received an equivalent dose of inactivated ERA-2GnRH in a chitosan hydrogel (Hydro). The end of the study (E) was approximately 70 days post-vaccination but varied for each group based on fertility.

Figure 3

Antibody dynamics of GnRH in mice vaccinated with the liquid ERA-2GnRH vaccine. Protein detection using the ProteinSimple Wes. (a) Detection of recombinant protein G-2GnRH using antibodies against GonaCon; lane 1, molecular weight (MW) marker; lane 2, recombinant G-2GnRH protein from purified ERA-2GnRH virus; (b) GnRH antibodies in representative mice receiving the liquid ERA-2GnRH vaccine over time; GnRH antibodies were detected in mouse 26 and 27 (Group B) from day 13 through day 41 and then decreased until the end of the experiment (E, ~day 70). Similarly, the rVNA titer was positive on day 13, went up on days 27 and 41, and declined until E.

Figure 4

GnRH antibodies in mice immunized with ERA-2GnRH hydrogel. The GnRH antibodies were delayed and detected from day 41 through the termination of the experiment around day 70, which is suggestive of a slow, controlled vaccine release profile.

Figure 5

Mechanism of GnRH-based antibodies on contraception. GnRH neurons in hypothalamus secrete GnRH to the hypophyseal portal system (HPS). Free GnRH in the HPS stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the anterior pituitary. After crossing the blood brain barrier, the LH and FSH work on the gonads and stimulate the reproduction cycle. The GnRH antibodies after vaccination with ERA-2GnRH bypass the Blood Brain Barrier through the HPS and catch/neutralize free GnRH in the median eminence, thus blocking the downstream signaling for reproduction.