Original
Article
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*
ORCID: https://orcid.org/0000-0003-2550-8807
Doaa I.
Rady ORCID: https://orcid.org/0000-0001-8877-5319
Eman R.
Abdo ORCID:
https://orcid.org/0000-0001-7710-5415
Alaa A.
El-Kholy ORCID: https://orcid.org/0000-0003-1016-727X
Veterinary
Sera and Vaccines Research Institute (VSVRI), Agriculture Research Center
(ARC), P.O.Box 131, Abbassia, Cairo 11381, Egypt.
Corresponding
author: aytollah@hotmail.com
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 disease; sheep pox virus; vaccination; cellular immune
response; ELISA.
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.
Submitted: October 27, 2021
Approved: December 16, 2021
Introduction
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 losses(3).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.
Materials and
Methods
Ethical
approval
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.
Cell
culture and virus
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)
Titration
of SPV vaccine on Vero cell line
The prepared live SPV
vaccines using Carbopol® and lactalbumin hydrolysate/sucrose was
titrated on Vero cell line.(1)
Control
sera
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.
Preparation
of carbomers
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.
Calves
and vaccination
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.
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)
Quality
control testing of the prepared SPV vaccine formulations
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.
Evaluation of humoral
immune response
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.
Results
Determination of the best concentration of
Carbopol® added to SPV vaccines
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).
Table 1. Mean NI and S/P (%) ratio
in calves immunized with SPV vaccines stabilized with different concentrations of Carbopol®.
|
|
Group 1 |
Group 2 |
Group 3 |
Group 4 |
Group 5 |
|||||
|
WPV |
NI |
S/P(%) |
NI |
S/P(%) |
NI |
S/P(%) |
NI |
S/P(%) |
NI |
S/P(%) |
|
0 |
0.50 |
5 |
0.50 |
5 |
0.50 |
5 |
0.50 |
5 |
0.50 |
5 |
|
1WPV |
1.00 |
15 |
1.00 |
17 |
1.25 |
19 |
1.00 |
17 |
0.25 |
5 |
|
2WPV |
1.75 |
32 |
1.75 |
35 |
2.00 |
35 |
1.75 |
35 |
0.25 |
5 |
|
3WPV |
2.25 |
*41 |
2.75 |
*45 |
2.50 |
*47 |
2.25 |
*41 |
0.25 |
5 |
|
4WPV |
*2.50 |
38 |
*3.00 |
42 |
*3.0 |
41 |
*2.50 |
38 |
0.25 |
5 |
|
5WPV |
2.50 |
37 |
2.75 |
45 |
2.75 |
45 |
2.50 |
39 |
0.25 |
5 |
|
6WPV |
2.50 |
35 |
2.75 |
44 |
3.0 |
45 |
2.50 |
36 |
0.25 |
5 |
WPV: weeks post vaccination.NI = Neutralization
index (NI ≥ 1.5 protective). S/P = sample to positive ratio (S/P >30
protective). *: peak of the antibody immune response. Group (1): calves
vaccinated with 0.25% Carbopol® stabilized SPV vaccine. Group (2): calves
vaccinated with 0.5% Carbopol® stabilized SPV vaccine. Group (3): calves
vaccinated with 1% Carbopol® stabilized SPV vaccine. Group (4): calves
vaccinated with lactalbumin hydrolysate/sucrose stabilized SPV vaccine. Group
(5): control non-vaccinated calves.
Quality control of prepared
live attenuated Carbopol® stabilized SPV vaccines
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.
Fig. 1. Final freeze-dried vials
for the SPV vaccine stabilized with 0.5% Carbopol® are capped with orange flip-off caps on the right side; whereas, the SPV
vaccine stabilized with lactalbumin hydrolysate/sucrose are green capped on the
left side.
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).
Table 2. SPV vaccines titers before and after
lyophilization.
|
SPV stabilized vaccine |
Virus titer log10TCID50/mL |
|
|
Before lyophilization |
After lyophilization |
|
|
SPV vaccine
without stabilizer |
5.5 |
- |
|
SPV vaccine
with 0.25 Carbopol® |
5.5 |
5.25 |
|
SPV vaccine with 0.5% Carbopol® |
5.5 |
5.25 |
|
SPV vaccine with 1% Carbopol® |
5.5 |
5.25 |
|
SPV vaccine with 5% lactalbumin hydrolysate
and 2.5% sucrose |
5.5 |
5.0 |
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.
Table 3. Thermo stability of the
prepared SPV vaccines.
|
Time of titration |
Virus titer (log10TCID50/mL) |
|||
|
SPV 10.5.5 TCID50/mL |
Reduction in virus
titer |
|||
|
0.5% Carbopol® |
Lactalbumin
hydrolysate/sucrose |
0.5% Carbopol® |
Lactalbumin
hydrolysate/sucrose |
|
|
Before
lyophilization |
5.5 |
5.5 |
0.0 |
0.0 |
|
After
lyophilization |
5.25 |
5.0 |
0.25 |
0.5 |
|
1st DPL* |
5.25 |
5.0 |
0.25 |
0.5 |
|
2nd DPL |
5.0 |
4.75 |
0.5 |
0.75 |
|
3rd DPL |
5.0 |
4.75 |
0.5 |
0.75 |
|
4th DPL |
4.5 |
4.25 |
1.0 |
1.25 |
|
5th DPL |
4.5 |
4.0 |
1.0 |
1.5 |
|
6th DPL |
4.25 |
3.75 |
1.25 |
1.75 |
|
7th DPL |
3.5 |
3.00 |
2.0 |
2.5 |
*DPL: days post
lyophilization
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.
Table 4. Optical density (OD) of cell mediated immune response in calves
vaccinated with 0,5% and 1% Carbopol® and lactalbumin hydrolysate/sucrose SPV vaccines.
|
Calves’ groups |
Calf number |
OD of cell mediated immune response on DPV* |
||||||||
|
0 |
1DPV |
3DPV |
5DPV |
7DPV |
*10DPV |
14DPV |
21DPV |
28DPV |
||
|
Group 2 |
1 |
0.082 |
0.281 |
0.451 |
0.671 |
0.825 |
**1.853 |
1.654 |
0.831 |
0.413 |
|
2 |
0.089 |
0.271 |
0.471 |
0.692 |
0.862 |
**1.862 |
1.684 |
0.751 |
0.407 |
|
|
3 |
0.092 |
0.321 |
0.467 |
0.683 |
0.913 |
**1.852 |
1.623 |
0.837 |
0.475 |
|
|
Average |
0.087 |
0.291 |
0.463 |
0.682 |
0.866 |
**1.855 |
1.653 |
0.806 |
0.431 |
|
|
Group 3 |
1 |
0.079 |
0.287 |
0.462 |
0.685 |
0.883 |
**1.838 |
1.662 |
0.853 |
0.438 |
|
2 |
0.087 |
0.261 |
0.460 |
0.643 |
0.860 |
**1.819 |
1.609 |
0.743 |
0.401 |
|
|
3 |
0.083 |
0.323 |
0.485 |
0.717 |
0.893 |
**1.917 |
1.783 |
0.846 |
0.447 |
|
|
Average |
0.083 |
0.290 |
0.469 |
0.681 |
0.878 |
**1.858 |
1.684 |
0.814 |
0.428 |
|
|
Group 4 |
1 |
0.075 |
0.162 |
0.264 |
0.381 |
0.650 |
**1.490 |
0.953 |
0.751 |
0.362 |
|
2 |
0.065 |
0.093 |
0.247 |
0.395 |
0.618 |
**1.415 |
1.025 |
0.724 |
0.382 |
|
|
3 |
0.080 |
0.164 |
0.289 |
0.401 |
0.738 |
**1.596 |
1.184 |
0.764 |
0.319 |
|
|
Average |
0.073 |
0.139 |
0.266 |
0.373 |
0.668 |
**1.500 |
1.054 |
0.746 |
0.354 |
|
|
Control Group 5 |
1 |
0.084 |
0.081 |
0.087 |
0.085 |
0.082 |
0.083 |
0.086 |
0.084 |
0.087 |
|
2 |
0.081 |
0.079 |
0.085 |
0.083 |
0.081 |
0.085 |
0.087 |
0.081 |
0.083 |
|
|
3 |
0.073 |
0.078 |
0.74 |
0.081 |
0.076 |
0.084 |
0.079 |
0.085 |
0.080 |
|
|
Average |
0.079 |
0.079 |
0.082 |
0.083 |
0.079 |
0.084 |
0.081 |
0.083 |
0.083 |
|
DPV*: days post vaccination. **: highest OD.
Group 2: calves vaccinated with SPV vaccine stabilized with
0.5% Carbopol®. Group 3: calves
vaccinated with SPV vaccine stabilized with
1.0% Carbopol®. Group 4: calves
vaccinated with lactalbumin hydrolysate/sucrose SPV vaccine.
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.
Table 5. Mean NI and S/P ratio of
vaccinated calves with different doses of SPV vaccine.
|
WPV |
Grupo 6 NI S/P(%) |
Grupo 7 NI S/P(%) |
Grupo 8 NI S/P(%) |
Grupo 9 NI S/P(%) |
Grupo 5 NI S/P(%) |
|||||
|
0 |
0.50 |
5 |
0.50 |
5 |
0.50 |
5 |
0.50 |
5 |
0.50 |
5 |
|
1 |
1.00 |
16 |
1.25 |
18 |
1.00 |
17 |
1.25 |
20 |
0.25 |
5 |
|
2 |
2.00 |
33 |
1.75 |
34 |
1.75 |
35 |
1.75 |
38 |
0.25 |
5 |
|
3 |
2.50 |
*47 |
2.75 |
*48 |
2.25 |
*40 |
2.25 |
*42 |
0.25 |
5 |
|
4 |
*3.00 |
42 |
*3.0 |
41 |
*2.50 |
37 |
*2.50 |
38 |
0.25 |
5 |
|
5 |
2.75 |
46 |
2.75 |
47 |
2.25 |
38 |
2.50 |
36 |
0.25 |
5 |
|
6 |
2.75 |
44 |
3.0 |
45 |
2.50 |
36 |
2.50 |
37 |
0.25 |
5 |
WPV: weeks post vaccination. NI: neutralization
index (NI ≥ 1.5 protective). S/P: sample to positive ratio (S/P >30%
protective). *: peak antibody levels. Group 6: vaccinated with a dose of 103.5 TCID50/animal (Carbopol® stabilized SPV vaccine). Group
7: vaccinated with a dose of 104 TCID50/animal
(Carbopol® stabilized SPV vaccine). Group 8: vaccinated with a dose of 103.5TCID50/animal (lactalbumin stabilized SPV vaccine). Group 9:
vaccinated with a dose of 104 TCID50/animal
(lactalbumin
stabilized SPV vaccine). Group 5: non-vaccinated
group.
Discussion
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 obtained,(6) 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)
Conclusion
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.
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Conflict of interest
The authors
whose names are listed above certify that they have no affiliations with or
involvement in any organization or entity with any financial and nonfinancial
interest in the subject matter or materials discussed in this manuscript.
Contributors
Ayatollah. I. Ibrahim:
study design, sampling, Methodology, interpretation of results and writing the
manuscript.
Doaa I. Rady: study design,
sampling, methodology, interpretation of results and writing the manuscript.
Eman R. Abdo: study design,
sampling, methodology, interpretation of results and writing the manuscript.
Alaa A. El-Kholy: study
design, sampling, methodology, interpretation of results and writing the
manuscript.
All
authors approved the final version of the manuscript.
* Senior researcher, Veterinary Sera and
Vaccines Research Institute, Egypt.