ABSTRACT: In Egypt, the lyophilized live attenuated sheep pox virus vaccine has been used for the vaccination of cattle against lumpy skin disease virus to control its economic impact on livestock industry. In this endeavor, we validate the efficacy of Carbopol® as a stabilizer and adjuvant to enhance immunogenicity of such a heterologous sheep pox virus vaccine against lumpy skin disease. Lyophilization of sheep pox virus vaccine stabilized with Carbopol® produced better physical and antigenic properties than freeze-drying with lactalbumin/sucrose stabilizer; this was manifested by superior disc uniformity, thermo-stability at 37oC, and less reduction in virus titer. Immunization of calves' groups with variable sheep pox vaccine doses containing different Carbopol® concentrations revealed that 103.5 TCID50 of sheep pox virus vaccine enclosing 0.5% Carbopol® is the field dose of choice. Moreover, it induced protective serum neutralizing index of 2.5 and a ELISA S/P ratio of 36, by the 4th week post vaccination. Besides, the inclusion of 0.5% Carbopol® in formulation of the sheep pox virus vaccine was safe in bovines and enhanced cellular immune response to lumpy skin disease virus, as evidenced by increased T cell proliferation. Hence, it is recommended to use Carbopol® as 0.5% in preparation of live attenuated sheep pox virus vaccine to confer better protection against lumpy skin disease virus infection.
Keywords: lumpy skin diseaselumpy skin disease,sheep pox virussheep pox virus,vaccinationvaccination,cellular immune responsecellular immune response,ELISAELISA.
RESUMEN: En Egipto, la vacuna atenuada liofilizada contra el virus de la viruela ovina ha sido utilizado para la vacunación del ganado, contra el virus de la dermatosis nodular contagiosa, para controlar su impacto económico en la industria ganadera. En este trabajo, validamos la eficacia del Carbopol®, como estabilizador y adyuvante, para mejorar la inmunogenicidad de dicha vacuna heteróloga contra la dermatosis nodular contagiosa. La liofilización de la vacuna contra el virus de la viruela ovina estabilizada con Carbopol®, resultó en mejores propiedades físicas y antigénicas que la liofilización con el estabilizador de lactoalbúmina/sacarosa; lo anterior se manifestó en la uniformidad superior del disco, la termoestabilidad a 37°C y la menor reducción del título del virus. La inmunización de grupos de terneros con dosis variables de vacuna contra el virus de la viruela ovina, que contenían diferentes concentraciones de Carbopol®, reveló que la dosis de campo de elección fue 103,5 TCID50 de la vacuna contra el virus de la viruela ovina conteniendo 0,5% de Carbopol®, la que indujo un índice de neutralización sérica protectora de 2,5 y una relación S/P de ELISA de 36 a la cuarta semana después de la vacunación. Además, la inclusión de Carbopol® al 0,5% en la formulación de la vacuna contra el virus de la viruela ovina fue segura en los bovinos y potenció la respuesta inmunitaria celular contra el virus de la dermatosis nodular contagiosa, como lo demuestra el aumento de la proliferación de células T. Por lo tanto, se recomienda el uso de Carbopol® al 0,5% en la preparación de la vacuna viva atenuada contra el virus de la viruela ovina para conferir una mejor protección contra la infección por el virus de la dermatosis nodular contagiosa.
Palabras clave: dermatosis nodular contagiosa, virus de la viruela de la oveja, vacunación, inmunidad celular, ELISA.
Artículo Original
Comparative efficacy of different Carbopol concentrations as a stabilizer of live attenuated sheep pox virus vaccine against lumpy skin disease
Eficacia comparativa de diferentes concentraciones de Carbopol como estabilizador de la vacuna viva atenuada contra el virus de la viruela ovina contra la dermatosis nodular contagiosa
Ayatollah I. Ibrahim
Doaa I. Rady
Eman R. Abdo
Alaa A. El-Kholy
Received: 27 October 2021
Accepted: 16 December 2021
Lumpy skin disease (LSD) is an acute infectious disease of cattle and has become endemic in Egypt and other African and Asian countries, exerting a serious economic impact on livestock industry. It is caused by LSD virus, a member of the Capripoxvirus genus and the Poxviridae family, along with the caprine pox virus (GPV) and the ovine pox virus (SPV).1,2 In Egypt, LSD was reported in 1988, at the Suez quarantine station and reappeared during 2005, 2006, 2012, 2013 and 2018, with significant economic losses3).Clinical signs include fever, cutaneous nodules on different regions of animal body and lesions in mucous membranes of the eyes, mouth and nose. Additionally, LSD causes reduced milk production, weight loss, abortions, inconsumable meat and skin, and death, especially in young calves.3
LSD virus (LSDV), SPV and GPV are closely antigenically and genetically related, share 97% of genomic nucleotide sequence identity and induce a reasonable cross-protection.4 Therefore, use of a capri pox virus vaccine strain (for example, SPV vaccine) can protect cattle against LSD and sheep and goats against sheep pox virus and goat pox virus diseases, respectively.5 In Egypt (unlike other African countries that have used the homologous Neethling LSD strain vaccine), the Kenyan SPV vaccine was used during 2005-2006. Currently, the Romanian SPV strain vaccine is used to immunize cattle against LSD. It provides a partial tackle for LSD outbreaks with partial cross protection and short-lived immunity. After the recent LSD outbreaks, the use of the SPV vaccine has shown inadequate protection against LSD in cattle.6)
The development of effective vaccines is an approach to provide cost-effective intervention against zoonotic and animal infectious diseases. In regions with a given enzootic disease, vaccination of the susceptible animal host is the milestone to control such a disease. Vaccines, whether live or inactivated, require formulations with adjuvants that act as vehicles or immunostimulants. Carbomers are synthetic, high molecular weight, nonlinear polymers of acrylic acid, cross-linked with a polyalkenyl polyether. These polymers are safely used in pharmaceutical and cosmetic preparations as thickening, suspending, dispersing, emulsifying or stabilizing mediators with different concentrations (0.1% - 50%), mostly below 1%.7,8).
Carbomers have been used as adjuvants in veterinary vaccines since 1970s, as they facilitate vaccine delivery and enhance cellular immune response with prolonged duration whether live or inactivated, such as: live-attenuated Newcastle disease virus (NDV) vaccine; modified-live vaccine for porcine reproductive and respiratory syndrome (PRRS1); swine parvovirus vaccine; circovirus type 2 vaccine; Staphylococcus aureus vaccine for sheep; freeze-dried inactivated bovine viral respiratory combined vaccine; inactivated equine herpes cirus-1 vaccine; inactivated rabies vaccine and freeze-dried bovine ephemeral fever virus vaccine.9,10,11,12,13 These studies proved the safe use of carbomers in vaccine preparation and stronger immune responses than those obtained by traditional vaccines. Carbopol® enhances cellular immunity by targeting a strong type-1 T-cell (Th1) polarization and induction of interferon-gamma (IFNγ) production. It also enhances antigen capture by macrophages, especially the dendritic cells.14,15
This study attempted to evaluate Carbomers to improve the stability of locally produced Romanian SPV vaccine and to enhance its protective efficacy as a heterologous vaccine against LSD infection in cattle.
This study was approved by the Animal Ethics Committee of the Veterinary Sera and Vaccines Research Institute (VSVRI). All experiments matched with the VSVRI guidelines for animal research.
African green monkey Kidney (Vero) cells were grown at 37ºC in Minimum Essential Medium with Earle's salts (MEM, Sigma Chemical Company, UK) supplemented with heat-inactivated 10% Newborn calf serum, 100 UI/mL penicillin,100 μg/mL streptomycin sulphate and 25 IU/L mycostatin (Gibco Laboratories, New Zealand).
The live attenuated sheep pox virus (SPV, Romanian strain), with a titer of 105.5 TCID50/mL, was kindly obtained from the Pox Vaccines Researches Department (PVRD), VSVRI, Abbassia, Cairo, Egypt.
The virus was propagated and titrated on Vero cell line which has been proved free of any extraneous contamination. Both virus and cell culture were used in vaccine preparations and virus neutralization test (VNT).1).
The prepared live SPV vaccines using Carbopol® and lactalbumin hydrolysate/sucrose was titrated on Vero cell line.1
Positive anti-SPV serum
Anti-SPV serum was prepared in three adult New Zealand rabbits (2-3 kg body weight), for use as a positive control serum in the VNT.1).
Negative serum
Antibody free Newborn calf serum (Gibco Laboratories, New Zealand) was used as negative control serum in VNT.
The carbomer used in vaccine preparations was Carbopol® 940 NF polymer (Lubrizol®). It was dissolved in hot double-distilled water at final concentrations of 0.25%, 0.5% and 1%, then solutions were sterilized separately by autoclaving at 121°C for 20 min and stored at 4°C. Before use in vaccine preparation, all Carbopol® solutions were adjusted to pH 7.3.
Thirty susceptible native breed calves 4-12 months old were assigned into nine groups, three calves each. They were proved free from virus neutralizing antibody against LSDV by VNT using SPV.
All animals were housed in hygienic, insect proof isolated units, at VSVRI animal facility, with daily clinical observation for 2 weeks before and during the experimentation period. Heparinized whole blood and serum samples were collected post vaccination (PV) at pre-scheduled intervals. Sera were stored at -20°C until testing by ELISA and VNT to evaluate the immune response to the prepared SPV vaccine formulations.
Sheep pox virus, titer105.5 TCID50/mL, was inoculated into Vero cell line and harvested after 4 days post inoculation when monolayers showed 70% cytopathic effect. After three freeze-thaw cycles, centrifugation was carried out and the supernatant was collected. Then two freeze-dried SPV vaccine formulations were prepared using different stabilizers as follows:
- Formula 1 was stabilized by the ordinary stabilizer consisting of a 5% lactalbumin hydrolysate (Sigma-Aldrich GmbH) with a 2.5% sucrose (Difco Laboratories, USA).1
- Formulas 2, 3 and 4 were stabilized by 0.25%, 0.5% and 1% carbomers (Carbopol® 940 NF, Lubrizol, USA), respectively.10,11,13
Physical appearance
All lyophilized SPV vaccine formulations containing Carbopol® and lactalbumin hydrolysate/sucrose stabilizer underwent physical examination.
Thermo stability test
Samples of the prepared SPV vaccine, stabilized with the Carbopol® concentration, selected as the best, were kept at 37˚C for one week and subjected to virus titration every 24 hour.16
Sterility
Random samples from the prepared SPV vaccines stabilized with Carbopol® were tested for their freedom from aerobic and anaerobic bacteria, fungi and mycoplasma on thioglycolate medium, Sabouraud agar, nutrient agar and mycoplasma medium.1
Safety
A group of three susceptible calves (Group 10) were vaccinated with 10X Carbopol® stabilized SPV vaccine field dose.1
Calves immunization with carbomers-stabilized SPV vaccines
Calves (groups 1- 4) were vaccinated with the prepared Carbopol®-stabilized SPV vaccine formulations, as follows:
Group 1: vaccinated with SPV vaccine stabilized with 0.25% Carbopol®.
Group 2: vaccinated with SPV vaccine stabilized with 0.5% Carbopol®.
Group 3: vaccinated with SPV vaccine stabilized with 1% Carbopol®.
Group 4: vaccinated with SPV vaccine stabilized with 5% lactalbumin hydrolysate/2.5% sucrose.
Group 5: non-vaccinated control.
Serum samples were taken weekly post vaccination up to 6 weeks for VNT and ELISA from all calves’ groups.
Virus neutralization test
VNT was conducted to estimate the seroconversion (antibody titers) in sera from vaccinated calves, as well as to determine the virus neutralization indices (NI) which were calculated by the following equation: NI = Virus Titer (VT)- Serum Virus Titer (SVT), where NI ˃1.5 were consider positive results.1,6
Commercial ELISA kit
A commercial ELISA kitID Screen® Capripox Double Antigen Multi-species, manufactured by IDvet (France) ID Vet., batch /N*de lot H35, was used to screen serum samples collected from the different calves’groups. The ELISA test was done according to manufacturer’s instructions. The results were interpreted based on the calculated S/P ratio.
If the S/P ratio is higher than 30%, the tested sample is considered a positive result for the presence of LSDV specific antibodies.
Cell proliferation assay
Collected heparinized blood samples at days 3, 5, 7, 10, 14, 21 and 28 post vaccination from all vaccinated and non-vaccinated calves, were used for the cell proliferation assay that was performed using the cell proliferation kit (XTT, Cat. No.11465017005, Sigma-Aldrich). Optical density (OD) was measured using an ELISA reader.
Determination of the optimal field dose of SPV vaccines
The optimum field protective dose of SPV vaccine was verified by vaccinating calves (groups 6-9) intradermally, with different potential doses, as shown hereafter:
Group 6: vaccinated with a dose of 103.5 TCID50/animal of Carbopol® stabilized SPV vaccine.
Group 7: vaccinated with a dose of 104 TCID50/animal of Carbopol® stabilized SPV vaccine.
Group 8: vaccinated with a dose of 103.5 TCID50/animal of lactalbumin hydrolysate/sucrose stabilized SPV vaccine.
Group 9: vaccinated with a dose of 104 TCID50/animal of lactalbumin hydrolysate/sucrose stabilized SPV vaccine.
Serum samples were taken weekly post vaccination up to 6 weeks for VNT and ELISA from all calves’ groups.
The mean NI and S/P ratio obtained for the SPV vaccines stabilized with different concentrations of Carbopol® are tabulated in Table 1. Protective levels of antibodies were induced in all calves immunized with vaccines containing different concentrations of Carbopol® (groups 1, 2 and 3), as well as in calves immunized with the lactalbumin hydrolysate/sucrose stabilized vaccine (group 4). Non-protective antibodies were induced in control calves (group 5). The highest levels of antibodies were detected in groups 2 and 3, which showed the highest NI value (3, for both groups) and similar S/P values (45%, 47% respectively); while in calves immunized with the lactalbumin hydrolysate/sucrose stabilized vaccine (group 4), the NI value was 2.50 and S/P=41%. Therefore, 0.5% Carbopol® was the concentration of choice for using with SPV vaccine (103.5TCID50/field dose).
Physical appearance
As shown in Figure 1, the lyophilized vaccine prepared with lactalbumin hydrolysate/sucrose stabilizer alone was yellowish, uniform in shape, and somewhat adherent to the vial walls with some vacuolation in the vaccine disk, in contrast, the lyophilized vaccine formulations with 0.5% Carbopol® exhibited better physical properties, the disk remained situated within the walls of the vials as whitish uniform compact solid disks.
Titration of prepared SPV vaccines with different concentrations of Carbopol® before and after lyophilization
Stabilized SPV vaccines prepared with different concentrations of Carbopol® showed the same reduction in virus titer (0.25 log10TCID50/mL) after lyophilization, while reduction in titer was higher (0.5 log10TCID50/mL) when lactalbumin/hydrolysate sucrose stabilizer was used (Table 2).
Thermo stability tests of the prepared vaccines
Thermo stability of the prepared lyophilized SPV vaccine with 0.5% Carbopol® (concentration of choice for use in SPV vaccine) and lactalbumin hydrolysate/sucrose stabilizer kept at 37˚C for one week, showed reduction in virus titer as 2log10TCID50/mL and 2.5log10 TCID50/mL, respectively, as shown in Table 3.
Sterility test
All prepared SPV vaccine stabilized with Carbopol® and lactalbumin hydrolysate/sucrose were proved to be free from any bacterial, fungal and mycoplasma contamination.
Safety test
Inoculation of 10X Carbopol® stabilized SPV vaccine in calves proved the prepared vaccine was safe to be used in calves, since the vaccinated calves did not show systemic or local reactions.
Cell mediated immune response of calves vaccinated with Carbopol® and lactalbumin hydrolysate/sucrose stabilized SPV vaccines
The highest level of cell proliferation was determined at day 10 in all vaccinated groups. Increasing mean OD values (1.855 and 1.858) were observed in groups 2 and 3 vaccinated with 0.5 and 1% Carbopol® stabilized SPV vaccines, respectively. Mean OD 1.500 was obtained for calves vaccinated with lactalbumin hydrolysate/sucrose stabilized SPV vaccine (group 4), while low OD (that not exceed 0.087) was read in control non-vaccinated (group 5), as shown in Table 4.
Determination of the optimal dose of prepared SPV vaccine
The results of mean NI and S/P ratio obtained for different doses of SPV vaccines are shown in Table 5. Protective levels of antibodies (NI=3 and S/P ratio (47% and 48%) were induced in calves vaccinated with 103.5 TCID50 and 104 TCID50 0.5% carbomer stabilized SPV vaccines (groups 6, 7), respectively; while in calves vaccinated with the 103.5TCID50 and 104TCID50 lactalbumin hydrolysate/sucrose stabilized SPV vaccines (groups 8 and 9) the detected NI value was 2.5, in both groups and the S/P ratio was 40% and 42%, respectively. The optimal dose of choice of the SPV vaccine was 103.5 TCID50/animal.
LSD is a disease of economic importance that affects cattle. The use of heterologous vaccination with SPV vaccine is a LSDV infection control strategy. An incomplete protection with SPV vaccine is obtained6, then the use of Carbopol® as adjuvant stabilizer can improve such heterologous vaccination.
The highest level of antibodies detected by NI and S/P in calves vaccinated with different concentrations of Carbopol® were obtained in groups 2 and 3 that received 0.5% and 1% Carbopol® stabilized SPV vaccine. Therefore, 0.5% carbomer was the concentration of choice to be used with SPV vaccine (103.5TCID50/field dose). Similar results were obtained for different animal viral vaccines like lyophilized live-attenuated NDV vaccine LaSota, freeze-dried inactivated bovine viral respiratory combined vaccine, modified-life vaccine Ingelvac PRRS1 MLV, tissue culture inactivated rabies vaccine, inactivated equine herpes virus-1 and freeze-dried bovine ephemeral fever virus vaccine.9,10,11,12,13
The vaccine stabilizer is a corner stone in maintaining the vaccine efficacy specially when exposed to high temperature; it plays an important role in prolongation of the shelf life of vaccine. The lyophilized Carbopol® SPV vaccine showed several advantages than the usual prepared lactalbumin hydrolysate/sucrose SPV vaccine, since it exhibited better physical properties and formation of whitish uniform compact solid disks on contrast to the yellowish, vacuolated contracted disk in case of using lactalbumin hydrolysate/sucrose stabilizer. It can be explained on the basis that the Carbopol® polymer is able to retain and absorb water to form agglomerates of polymer chains that are irreversible,8,17 and makes a high uniformity white compact disk of prepared NDV vaccine when carbomer is used in 0.5% concentration, in comparison to the skimmed milk stabilizer.10).
In addition to the physical properties, the prepared Carbopol® stabilized vaccine maintained a higher virus titer after lyophilization (105.25TCID50/mL) compared to lyophilized lactalbumin hydrolysate/sucrose SPV vaccine (105.0TCID50/mL); in addition, minor virus reduction was obtained when exposed at 37°C for one week in thermo stability test, since the decrease in virus titer for Carbopol® stabilized SPV vaccine was 2 log10TCID50/mL, while in case of SPV vaccine prepared by using lactalbumin hydrolysate/sucrose stabilizer, was 2.5 log10TCID50/mL. Similar results were obtained by the prepared freeze-dried bovine viral respiratory combined vaccine using 0.5% carbomer as stabilizer. Carbomer binds to virus particles and forms a tight sealed that protects the integrity of the formed complex, even after heat exposure, keeping the thermo stability properties of the prepared vaccine.13
The testing of the prepared SPV vaccine stabilized with Carbopol® indicated its freedom of fungal and bacterial contamination. Furthermore, none of the vaccinated calves with the 10x field dose experienced any systemic or local reactions or mortality; this agreed with the information provided in the Carbopol® safety data sheet and indicates the non-toxic properties of carbomer when used as a stabilizer.7
The immune response to capripox vaccination and infection is mainly cell mediated. One the most important components in capripox cellular immune response in dermal layer are dendritic cells, which are characterized by induction of IFN-γ.18). Due to biosafety requirements, the cellular immune response for the prepared SPV vaccine was measured, instead of challenge test. The highest values of cell proliferation were detected at 10th day PV in all vaccinated groups, with an increase in mean OD values (1.855 and 1.858) in calves vaccinated with 0.5 and 1% Carbopol® stabilized SPV vaccines (group 6 and 7), respectively, compared to group 8 (vaccinated with the lactalbumin hydrolysate/sucrose SPV vaccine) that showed a mean OD 1.500, showing the great effect of Carbopol® on cellular immunity. This carbomer stimulates CD8 T-cell through induction of a different metabolic state in dendritic cells that produce more IL-1β and IL-18; also Carbopol® induces early IFNγ which stimulates T-cell differentiation into specific effector phenotypes.(7, 9,14)
Serum samples collected from calves vaccinated with different field doses of the SPV vaccine (103.5 and 104 TCID50/field dose) stabilized with Carbopol® (groups 6 and 7) and lactalbumin/hydrolyzate sucrose (groups 8 and 9), were evaluated based on serological tests. The results of the mean NI and S / P ratios showed protective antibody levels in all vaccinated calves. Regarding to the mean NI and S/P obtained for 103.5 and 104 TCID50/field doses, the dose of choice for the SPV vaccine was 103.5 TCID50/field dose; this result corresponds with the recommendation that a 10X field dose of SPV vaccine used for sheep against SPV, develops sufficient protection against LSD in cattle.4,5,19
The use of Carbopol® not only enhanced the physical properties of the prepared lyophilized SPV vaccines, but it also increased and improved the humoral and cellular immune response of vaccinated calves and could overcome the incomplete protection of heterologous vaccination with SPV vaccine against LSD.
Corresponding author: aytollah@hotmail.com
