Development of a Safe and Highly Efficient Inactivated Vaccine Candidate against Lumpy Skin Disease Virus

Abstract

Capripox virus (CaPV)-induced diseases (lumpy skin disease, sheeppox, goatpox) are described as the most serious pox diseases of livestock animals, and therefore are listed as notifiable diseases under guidelines of the World Organisation for Animal Health (OIE). Until now, only live-attenuated vaccines are commercially available for the control of CaPV. Due to numerous potential problems after vaccination (e.g., loss of the disease-free status of the respective country, the possibility of vaccine virus shedding and transmission as well as the risk of recombination with field strains during natural outbreaks), the use of these vaccines must be considered carefully and is not recommended in CaPV-free countries. Therefore, innocuous and efficacious inactivated vaccines against CaPV would provide a great tool for control of these diseases. Unfortunately, most inactivated Capripox vaccines were reported as insufficient and protection seemed to be only short-lived. Nevertheless, a few studies dealing with inactivated vaccines against CaPV are published, giving evidence for good clinical protection against CaPV-infections. In our studies, a low molecular weight copolymer-adjuvanted vaccine formulation was able to induce sterile immunity in the respective animals after severe challenge infection. Our findings strongly support the possibility of useful inactivated vaccines against CaPV-infections, and indicate a marked impact of the chosen adjuvant for the level of protection.

Keywords: LSDV; adjuvants; capripox; inactivated vaccine; lumpy skin disease; vaccine.

Figure 1

Experimental design of the performed proof-of-concept study. Cattle of Group 1A were not vaccinated. Cattle of Group 1B received the commercially available Herbivac LS live-attenuated vaccine at Day 14 of the animal trial. Cattle of Group 1C were vaccinated twice (Day 0 and Day 21 of the animal trial) with inactivated LSDV-“Neethling vaccine“ strain (titer before inactivation 10^7.4 CCID₅₀/mL) propagated on MDBK cells and using Adjuvant A. Challenge virus was inoculated i.v. and s.c. at Day 35 of the animal trial. Grey marks display sampling days.

Figure 2

Experimental design of the performed vaccine-efficacy study. Cattle of Group 2A were not vaccinated but received PBS instead. The cattle of the other groups were vaccinated with inactivated LSDV-“Serbia“ field strain propagated on a non-bovine cell line. Group 2B received a vaccine with a virus infectious titer before inactivation of 10⁷ CCID₅₀/mL and Adjuvant A. For Group 2C and 2D, the inactivated virus was used with different titers (10⁷ CCID₅₀/mL and 10⁶ CCID₅₀/mL, respectively) and Adjuvant B. Grey marks display sampling days.

Figure 3

Clinical reaction score of the cattle during the proof-of-concept animal trial. Clinical reaction score was measured daily starting a few days before first vaccination with inactivated LSDV until 28 dpc. (A) Cattle of Group 1A were left unvaccinated and served as challenge control group. (B) Cattle of Group 1B received the commercially available life-attenuated vaccine “Herbivac LS”, and (C) cattle of Group 1C were vaccinated with an inactivated vaccine prototype on basis of LSDV-“Neethling vaccine” strain.

Figure 3

Clinical reaction score of the cattle during the proof-of-concept animal trial. Clinical reaction score was measured daily starting a few days before first vaccination with inactivated LSDV until 28 dpc. (A) Cattle of Group 1A were left unvaccinated and served as challenge control group. (B) Cattle of Group 1B received the commercially available life-attenuated vaccine “Herbivac LS”, and (C) cattle of Group 1C were vaccinated with an inactivated vaccine prototype on basis of LSDV-“Neethling vaccine” strain.

Figure 4

Summarized presentation of adverse reactions after immunization, clinical reaction, viral genome load in different samples matrices and serological examination of the proof-of-concept-study. Adverse reactions are shown at representative days post vaccination. Furthermore, strength of clinical reaction and rise in body temperature after immunization or challenge infection are presented (left squares of each day) for each animal. Additionally, extend of viremia and shedding of viral genome as well as serological data (right squares of each day) following challenge infection are shown for each individual.

Figure 5

Viral genome load in (A–C) EDTA-blood and (D–F) nasal swab samples taken during the proof-of-concept study. (A+D) Cattle of Group 1A were left unvaccinated and served as challenge control group. (B + E) Cattle of Group 1B received the commercially available life-attenuated vaccine “Herbivac LS”, and (C + F) cattle of Group 1C were vaccinated with an inactivated vaccine prototype on basis of LSDV-“Neethling vaccine” strain. Samples were taken at defined time points during the animal trial and analyzed regarding their viral genome load. Cut-off was defined at Cq 40.0.

Figure 6

Seroconversion of the cattle of the proof-of-concept animal trial. Serum samples taken at certain time points during the study were analyzed using (A–C) the serum neutralization test (SNT) and (D–F) the DA ELISA. (A + D) Cattle of Group 1A were left unvaccinated and served as challenge control group. (B + E) Cattle of Group 1B received the commercially available life-attenuated vaccine “Herbivac LS”, and (C + F) cattle of Group 1C were vaccinated with an inactivated vaccine prototype on basis of LSDV-“Neethling vaccine” strain. Samples in the SNT were defined positive at ND₅₀/mL ≥ 13, whereas in the ELISA samples with S/P% ≥ 30 are positive.

Figure 6

Seroconversion of the cattle of the proof-of-concept animal trial. Serum samples taken at certain time points during the study were analyzed using (A–C) the serum neutralization test (SNT) and (D–F) the DA ELISA. (A + D) Cattle of Group 1A were left unvaccinated and served as challenge control group. (B + E) Cattle of Group 1B received the commercially available life-attenuated vaccine “Herbivac LS”, and (C + F) cattle of Group 1C were vaccinated with an inactivated vaccine prototype on basis of LSDV-“Neethling vaccine” strain. Samples in the SNT were defined positive at ND₅₀/mL ≥ 13, whereas in the ELISA samples with S/P% ≥ 30 are positive.

Figure 7

Summarized presentation of adverse reactions after immunization, clinical reaction, viral genome load in different samples matrices and serological examination of the vaccine-efficacy study. A rough overview of adverse reactions after immunization is shown. Furthermore, strength of clinical reaction and rise in body temperature after immunization or challenge infection are presented (left squares of each day) for each animal. Additionally, extent of viremia and shedding of viral genome as well as serological data (right squares of each day) following challenge infection are shown for each individual.

Figure 8

Clinical reaction score of the cattle during the vaccine-efficacy study. (A) Cattle of Group 2A serve as mock-control and received PBS. Animals of the other groups were immunized with inactivated LSDV-“Serbia” field strain using different adjuvants and virus titers before inactivation. (B) Cattle of Group 2B were vaccinated with Adjuvant A 10⁷, (C) cattle of Group 2C received Adjuvant B 10⁷, and (D) animals of Group 2D were immunized with Adjuvant B 10⁶ prototype vaccine. Clinical reaction score was calculated daily from the day of challenge infection until 28 dpc.

Figure 9

Comparison of skin nodules observed after challenge infection during the vaccine-efficacy study. After challenge infection, skin alterations occurred in some cattle previously immunized with Adjuvant B-containing vaccine. Although affected cattle of the challenge control group showed skin lesions typical for LSDV (e.g., R/783), skin alterations in cattle previously vaccinated with Adjuvant B 10⁷ (e.g., R/464) and Adjuvant B 10⁶ (e.g., R/833), respectively, were clearly different in shape and appearance (blue arrows).

Figure 10

Virus isolation from skin samples obtained from skin nodules of Adjuvant B-vaccinated cattle that were positive for Capripox virus genome. Skin samples were homogenized and incubated on susceptible MDBK cells for 7 days. Afterwards, cytopathic effect was analyzed using a light microscope. Virus isolation was successful of all skin samples that were tested positive for Capripox virus genome.

Figure 11

Viral genome load in (A–D) EDTA-blood and (E–H) nasal swabs taken during the vaccine-efficacy animal trial. (A+E) Cattle of Group 2A serve as mock-control and received PBS. Animals of the other groups were immunized with inactivated LSDV-“Serbia” field strain using different adjuvants and virus titers before inactivation. (B + F) Cattle of Group 2B were vaccinated with Adjuvant A 10⁷, (C + G) cattle of Group 2C received Adjuvant B 10⁷, and (D + H) animals of Group 2D were immunized with Adjuvant B 10⁶ prototype vaccine. The samples were taken at certain time points during the animal trial and analyzed regarding the viral genome load. Cut-off was set at Cq 40.0.

Figure 12

Serological examination of the sera taken during the vaccine-efficacy study using the DA ELISA. (A) Cattle of Group 2A serve as mock-control and received PBS. Animals of the other groups were immunized with inactivated LSDV-“Serbia” field strain using different adjuvants and virus titers before inactivation. (B) Cattle of Group 2B were vaccinated with Adjuvant A 10⁷, (C) cattle of Group 2C received Adjuvant B 10⁷, and (D) animals of Group 2D were immunized with Adjuvant B 10⁶ prototype vaccine. Samples were defined positive at an S/P% ratio ≥ 30.