Post-exposure Treatment with Anti-rabies VHH and Vaccine Significantly Improves Protection of Mice from Lethal Rabies Infection

Abstract

Post-exposure prophylaxis (PEP) against rabies infection consists of a combination of passive immunisation with plasma-derived human or equine immune globulins and active immunisation with vaccine delivered shortly after exposure. Since anti-rabies immune globulins are expensive and scarce, there is a need for cheaper alternatives that can be produced more consistently. Previously, we generated potent virus-neutralising VHH, also called Nanobodies, against the rabies glycoprotein that are effectively preventing lethal disease in an in vivo mouse model. The VHH domain is the smallest antigen-binding functional fragment of camelid heavy chain-only antibodies that can be manufactured in microbial expression systems. In the current study we evaluated the efficacy of half-life extended anti-rabies VHH in combination with vaccine for PEP in an intranasal rabies infection model in mice. The PEP combination therapy of systemic anti-rabies VHH and intramuscular vaccine significantly delayed the onset of disease compared to treatment with anti-rabies VHH alone, prolonged median survival time (35 versus 14 days) and decreased mortality (60% versus 19% survival rate), when treated 24 hours after rabies virus challenge. Vaccine alone was unable to rescue mice from lethal disease. As reported also for immune globulins, some interference of anti-rabies VHH with the antigenicity of the vaccine was observed, but this did not impede the synergistic effect. Post exposure treatment with vaccine and human anti-rabies immune globulins was unable to protect mice from lethal challenge. Anti-rabies VHH and vaccine act synergistically to protect mice after rabies virus exposure, which further validates the possible use of anti-rabies VHH for rabies PEP.

Fig 1. Rabies neutralizing activity in the blood as measured by RFFIT, following intramuscular (IM) vaccination at day -28 and day-25 either alone or in combination with anti-rabies VHH (Rab-E8-H7-ALB11, 1.5 mg/mouse) at day -28.

Control groups consisted of mock treatment with saline (0.9% NaCl) or anti-rabies VHH only at day -28. Blood was collected at day -28 (prior to vaccination and VHH administration), day -25, day -20 and day 0 (= time of virus challenge). Both groups of mice that received anti-rabies VHH had high neutralizing titers at the early time points (day -25 and -20). Mice that received vaccine had neutralizing antibodies at day -20, which further increased to high levels at day 0. Mice treated with anti-rabies VHH only no longer had detectable VHH at day 0. Antibody titers in the vaccine + VHH group were significantly lower than in the vaccine only group at day 0 (** p< 0.005, *** p< 0.001). Error bars represent the standard deviation.

Fig 2. Effect of pre-exposure vaccination on survival in rabies mouse model.

Mice received intramuscular (IM) vaccination at day -28 alone or in conjunction with intraperitoneal (IP) anti-rabies VHH (Rab-E8-H7-ALB11, 1.5 mg/mouse). Vaccinated mice received a booster vaccination at day -25. Control groups received a single dose of anti-rabies VHH or mock treatment (Saline) at day -28. Preventive vaccination could protect 50% of the animals from lethal infection whereas mice receiving vaccine simultaneously with anti-rabies VHH, or VHH alone, were significantly less protected from lethal disease (p<0.001).

Fig 3. Effect of post-exposure prophylactic treatment with vaccine and anti-rabies VHH on survival in rabies mouse model.

Mice were intranasally inoculated with rabies virus followed by treatment with anti-rabies VHH (IP) 24 hours later, either alone or in conjunction with vaccine (IM). Vaccinated mice received a second vaccine dose 3 days later. Control groups consisted of mice that were not treated (virus only group) or that received the vaccination regime only (vaccination group). Combined treatment with vaccine and anti-rabies VHH resulted in 60% survival, while treatment with anti-rabies VHH alone rescued 19% (p<0.01).

Fig 4. Effect of post-exposure prophylactic treatment with vaccine and human rabies immune globulins on survival in rabies mouse model.

Mice were intranasally inoculated with rabies virus followed by treatment with human rabies immune globulins (HRIG) (IP) 24 hours later, either alone or in conjunction with vaccine (IM). Vaccinated mice received a second vaccine dose 3 days later. The control group consisted of mice that were not treated (virus only group). Combined treatment with vaccine and human rabies immune globulins did not differ significantly from treatment with human rabies immune globulins alone and was unable to rescue mice from lethal infection.

Fig 5. Post-exposure treatment with vaccine and anti-rabies VHH: Effect on the viral RNA load in the brain of mice.

The viral load was determined at the peak of clinical symptoms in mice that developed disease (filled symbols) or at the end of the observation period (open symbols) in survivor (non-diseased) mice. The dashed line represents the limit of detection (= 5 ΔCq). Mice treated with vaccine + VHH had significantly lower viral RNA loads than naïve mice (p<0.0001), mice treated with vaccine only (p<0.001) or mice treated with VHH only (p<0.05). Viral loads of diseased mice were also lower (25.85 ΔCq) in mice that were treated with vaccine + VHH compared to naïve mice (29.58 ± 1.29 ΔCq) or treated with VHH only (29.59 ±0.76 ΔCq). Survivor mice (vaccine + VHH, VHH alone) had comparably low viral loads (3.3–11.3 ΔCq).