Evaluation of different adjuvant formulations of trivalent Foot and Mouth Disease vaccine in cattle

doi:10.21203/rs.3.rs-1975001/v1

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Evaluation of different adjuvant formulations of trivalent Foot and Mouth Disease vaccine in cattle

https://doi.org/10.21203/rs.3.rs-1975001/v1

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Background

Foot-and-mouth disease is globally one of the most economically important viral diseases of cloven-hoofed animals that can be controlled by different strategies, where vaccination plays an important role. Selection of the adjuvants,that added to the vaccine is crucial in ensuring the protective effect of the vaccine. Aluminum hydroxide gel and saponin (AS) is widely used adjuvant, with its poor immune response in FMD vaccine. The present study was undertaken to evaluate different ingredients of adjuvants for inactivated trivalent (A, O and SAT 2) FMD vaccine and to demonstrate the effect of booster dose in cattle.

Results

Cattle were grouped into five; four experimental and one control, with six animals in each group and immunized with trivalent vaccine with various formulations of adjuvants. Immune response was measured using Solid Phase Competitive Enzyme Linked Immune Sorbent Assay (SPCE). The antibody level in cattle immunised with a vaccine formulation containing a mixture of aluminum hydroxide gel and saponin (AS) were significantly lower than AS boosted group for the three serotypes (p < 0.05, t. test), which directs the need for booster dose. Whereas the antibody response in the AS + oil group was higher followed by oil alone, AS boosted and AS at 95% CI.

Conclusion

The findings of this study could suggest that oil based and AS with oil could replace the conventional aluminum hydroxide gel and saponin adjuvants in FMD vaccine preparations. On different note, challenge test was not successful in this study indicating the need for further research on the virus infectivity.

adjuvant

Foot and Mouth Disease

immune response

serotype

Ethiopia is one of the country which has the largest livestock population in Africa (1). Despite this, the products and productivity of livestock are still very less due to the impacts of economically important diseases of animals (2). Among these, foot-and- mouth disease (FMD) is highly contagious viral disease of cloven-hooved animals affecting cattle, pigs, sheep, and goats (3). This disease is characterized by fever and the presence of vesicular lesion or sores on the tongue, lips, teats, snouts, in the oral cavity and between the hooves. It is known by causing high morbidity, low mortality, severe production and weight losses in adult animals. However, it causes high mortality in young animals (4).

The FMD virus (FMDV) is the member of the genus Aphthovirus, which belongs to the family Picornaviridae. The virus genome is a positive single stranded RNA, which is about 8.5 Kb, encodes structural proteins including viral proteins (VP)1, VP2, VP3, VP4 and nonstructural proteins (5). To date, seven serotypes that are responsible to cause FMD have been identified such as Southern African Territories (SAT) 1, SAT 2, SAT 3, A, O, C, and Asia 1 with different genotypes/topotypes, lineages and variants (5,6). In Africa, six of the seven serotypes of FMD viruses except Asia 1 have been reported in the continent (7,8,46).Among these three serotypes, SAT 1, SAT 2 and SAT 3 are unique in Africa (9,10,46).

In Ethiopia, the disease is endemic and the report of Ministry of Agriculture showed that, the highest incidence of outbreak was occurred from 1997 to 2006 throughout the country. According to the report, the maximum outbreak was recorded in the central part of Ethiopia (11). Many sero-epidemiological surveys conducted in different parts of the country have showed a prevalence result of up to ~ 26%. Among the seven serotypes of FMDV that are immunologically different, five of them (O, A, SAT 1, SAT 2 and C) were reported from FMD samples collected throughout the country from 1981–2007(8,12,13,14). The FMDV serotype O is the most prevalent (73.3%) followed by serotype A (19.5%), SAT 2 (4.1%) and SAT 1 (1.8%). After 1983 serotype C has not been reported in Ethiopia (8,15,12).

The endemic presence of the FMDV is highly challenging due to its impacts on the trade market and productivity of the livestock. Thus, different measures are required to eradicate and control the disease, including vaccination strategies and control of animal movement (16). However, the vaccine requires continuous improvement to overcome the limitations of currently existing FMD vaccines and to increase its protective ability (17). This can be achieved by the selection of proper antigen and adjuvants mixed with the vaccine (18). An ideal adjuvant is the one which can stimulate specific components of humoral immune response early, that can promote production of high antibody titer with prolonged duration of immunity. It should also stimulate cell-based immune response (19).

In Ethiopia, considering the wide prevalence of the disease, National Veterinary Institute (NVI) is producing inactivated trivalent (for serotype O, A and SAT 2) vaccine. The vaccine is produced only for cattle and adjuvanted with aluminum hydroxide gel and saponin (20). Aluminum hydroxide gel and saponin and oil adjuvant vaccines are the most commonly used adjuvant in inactivated FMD vaccine to control the disease. The aluminum hydroxide gel and saponin adjuvant has an advantage in that the antigen is released slowly as it is absorbed into the gel. However, vaccine containing aluminum hydroxide gel and saponin as an adjuvant has several limitations, including the induction of short-lived immune responses and the consequent need for frequent vaccination. In contrast, the oil-adjuvanted FMD vaccines are effective in providing long lasting protection than a standard aluminum hydroxide gel and saponin based vaccines (21,22). Since 1970, adjuvants used in FMD vaccine have been transformed from gel to oil, thereby overcoming the problem of poor immunization (23). Comparative studies of aluminum hydroxide gel and saponin with oil-based adjuvant have been conducted and revealed that oil adjuvant showed better immune response than aluminum hydroxide gel and saponin (24). Providing early protection against the disease is another advantage of oil-based adjuvant (25). Furthermore, study conducted on combination of aluminum hydroxide gel and saponin and oil induced better immunity (26). Despite documented advantages of oil based and combined adjuvants in enhancing immune response, there is limited scientific evidence on adjuvant evaluation and selection of proper adjuvants in Ethiopia. Therefore, the present study was carried out to evaluate immune response induced by different formulation of adjuvants for inactivated trivalent FMD vaccines in cattle, assess immune response elicited with and without booster vaccine, and test the potency of formulated vaccines by challenging the experimental animals with the live virus.

Virus titration result

The infectivity titer calculated and expressed in log₁₀ of 50% tissue culture infectious dose (TCID₅₀) for the three serotypes of FMDV (A, O and SAT 2) using BHK-21 cell culture is listed in (Table 1).

Table 1

The infectivity dose of the viruses expressed in log₁₀ TCID₅₀ /ml
FMDV serotypes	Infectivity dose of the viruses in log₁₀
A	10⁷TCID₅₀ /ml
O	10^6.4TCID₅₀ /ml
SAT 2	10^7.2TCID₅₀ /ml

TCID = Tissue culture infectious dose

Sterility and safety test result

The sterility test of the vaccine formulations showed that the vaccines were free from aerobic and anaerobic bacteria, fungal and mycoplasma contaminants. Similarly, assessment of absence of CPE on BHK-21 cells was used to confirm complete virus inactivation (Table 2). Thus, the formulated vaccines were considered to be safe for animal experimentation as per the OIE requirements for vaccine preparation (27).

Table 2

Summary of sterility and safety test results of the prepared trivalent FMD vaccines
Bacteriological media/ cell culture used for the test	Vaccine formulations
Bacteriological media/ cell culture used for the test	AS	Oil	AS + oil
Thioglycolate broth (for bacterial contaminants)	-ve	-ve		-ve
Tryptone Soya broth (for bacterial contaminants)	-ve	-ve		-ve
Sabouraud agar (fungal contaminants)	-ve	-ve		-ve
Mycoplasma medium	-ve	-ve		-ve
BHK-21 cell culture	no CPE	no CPE		no CPE

-ve = Negative, BHK = Baby hamster kidney cell, CPE = Cytopathic effect, AS = Aluminum hydroxide gel and saponin

Immune response result for FMDV serotype A

In experimental groups inoculated with vaccine formulations containing a mixture of aluminum hydroxide gel and saponin, aluminum hydroxide gel and saponin with a booster dose and aluminum hydroxide gel, saponin and oil, and oil alone, there was an evident increase in antibody level from day 7 to 42 post vaccinations for serotype A (Table 3, Figs. 1 and 2). On day 42 after vaccination, the percent of inhibition observed was highest in experimental group where a mixture of aluminum hydroxide gel, saponin and oil was used as an adjuvant (PI = 99.26%) followed by groups inoculated with vaccine formulation containing oil alone (PI = 97.76%) and aluminum hydroxide gel and saponin with a booster dose (PI = 94.79%). The experimental group inoculated with formulations which contains aluminum hydroxide gel and saponin alone had the lowest immune response (PI = 89.57%) (Table 4). There was a significant difference in humoral immune response in experimental group vaccinated with formulation containing aluminum hydroxide gel and saponin mixed with oil (p < 0.05) as evidenced by the mean OD value and highest percentage of inhibition compared to other groups.

Table 3

Mean optical density (OD) of antibody response of cattle against FMDV serotype A
Days post vaccination
	Control (mean OD with CI)	AS (mean OD with CI)	AS boost (mean OD with CI)	Oil (mean OD with CI)	AS + oil (mean OD with CI)	P value
Day 0	1.72 (1.36- 2.0)	1.66 (1.14–2.17)	1.39 (0.84–1.94)	1.54 (1.22–1.86)	1.57 (1.17–1.96)	0.727
SE	0.13	0.20	0.21	0.12	0.15
Day 7	1.36 (0.96–1.75)	1.02 (0.89–1.13)	0.99 (0.70–1.27)	0.89 (0.71–1.08)	0.84 (0.71–0.97)	0.0052
SE	0.14	0.04	0.10	0.07	0.05
Day 14	1.48 (1.0-1.97)	1.48 (1.0-1.97)	0.82 (0.67–0.98)	0.63 (0.54–0.72)	0.44(0.35–0.51)	0.0000
SE	0.17	0.17	0.06	0.03	0.03
Day 21	1.1 (0.83–1.38)	0.62 (0.49–0.74)	0.43 (0.30–0.55)	0.32 (0.25–0.39)	0.16 (0.10–0.21)	0.0000
SE	0.09	0.04	0.04	0.02	0.02
Day 28	1.48 (1.04–1.92)	0.40 (0.28–0.52)	0.17 (0.09–0.24)	0. 07 (0.02–0.13)	0.03 (0.01–0.04)	0.0000
SE	0.15	0.04	0.02	0.02	0.01
Day 42	1.31 (1.09–1.53)	0.28 (0.15–0.41)	0.14 (0.06–0.20)	0.06 (0.02–0.11)	0.02 (0.01–0.02)	0.0000
SE	0.08	0.05	0.02	0.04	0.00
AS = Aluminum hydroxide gel and saponin, AS boost = Aluminum hydroxide gel and saponin boosted, AS + oil = Aluminum hydroxide gel, saponin and oil, CI = Confidence interval,OD = Optical density, SE = Standard error

Table 4

Mean percentage inhibition of antibody response of cattle against FMDV serotype A as measured by SPCE.
Adjuvant Formulation	Percentage of inhibition (PI%)
Adjuvant Formulation	Day 0	Day 7	Day 14	Day 21	Day 28	Day 42
Control	35.58	49.06	44.56	59.03	44.88	51.21
AS	37.82	61.79	70.03	76.9	85.1	89.57
AS boost	47.94	62.92	69.29	83.98	93.67	94.79
Oil	42.32	66.67	76.4	88.1	97.39	97.76
AS + oil	41.2	68.54	83.52	94.04	98.88	99.26
AS = Aluminum hydroxide gel and saponin, AS boost = Aluminum hydroxide gel and saponin boosted, AS + oil = Aluminum hydroxide gel, saponinand oil

Immune response result for FMDV serotype O

In all the four experimental groups the result revealed that the mean antibody response for FMDV serotype O increased considerably over all post vaccination period (Table 5, Figs. 3 and 4). Significantly higher antibody titers were recorded from day 0 to reach its maximum value at day 42 in experimental groups inoculated with formulations containing a mixture of aluminum hydroxide gel, saponin and mineral oil (1.58, 0.04), and oil alone (1.57, 0.05), (p < 0.05).However, the titers were significantly lower in experimental groups inoculated with vaccine preparation adjuvanted with a mixture of aluminum hydroxide gel and saponin (1.62, 0.19) followed by groups inoculated with mixture of aluminum hydroxide gel and saponin with a booster dose (1.44, 0.12) at day 0 and 42, respectively compared to other groups (p < 0.05). The maximum percent of inhibition at 42 days post vaccination was recorded in experimental groups inoculated with vaccine formulations containing a mixture of aluminum hydroxide gel, saponin and mineral oil (P = 98.47%) and mineral oil alone (P = 98.08%). The percent of inhibition in experimental group’s inoculated with vaccine formulations containing aluminum hydroxide gel and saponin with a booster dose was 95.4%, whereas this was 92.72% without a booster (Table 6).

Table 5

Mean optical density (OD) of antibody response of cattle against FMDV serotype O
Days post vaccination		Experimental groups
Days post vaccination		Control (mean OD with CI)	AS (mean OD with CI)	AS boost (mean OD with CI)	Oil (mean OD with CI)	AS + oil (mean OD with CI)	P value
Day 0	1.40 (0.8–1.99)		1.62 (1.08–2.16)	1.44 (0.69–2.19)	1.57 (1.09–2.04)	1.58 (1.02–2.14)	0.9544
SE	0.21		0.20	0.29	0.18	0.21
Day 7	1.36 (0.73–1.98)		1.06 (0.87–1.20)	0.86 (0.57–1.14)	0.86 (0.76–0.95)	0.93 (0.7–1.08)	0.0279
SE	0.07		0.07	0.11	0.03	0.06
Day 14	1.37 (1.1-31.61)		0.86 (0.81–0.93)	0.77 (0.50–1.04)	0.52 (0.47–0.61)	0.54 (0.39–0.69)	0.0000
SE	0.08		0.02	0.10	0.02	0.05
Day 21	1.18 (1.01–1.34)		0.62 (0.5–0.73)	0.45 (0.37–0.53)	0.31 (0.20–0.40)	0.16 (0.10–0.22)	0.0000
SE	0.05		0.04	0.03	0.04	0.02
Day 28	1.37 (1.19–1.55)		0.29 (0.22–0.37)	0.17 (0.06–0.26)	0.08 (0.00-0.15)	0.03 (0.01–0.03)	0.0000
SE	0.06		0.02	0.03	0.02	0.00
Day 42	1.42 (1.16–1.66)		0.19 (0.14–0.24)	0.12 (0.08–0.15)	0.05 (0.01–0.09)	0.04 (0.02–0.05)	0.0000
SE	0.08		0.01	0.01	0.04	0.01
AS = Aluminum hydroxide gel and saponin, AS boost = Aluminum hydroxide gel and saponin boosted, AS + oil = Aluminum hydroxide gel, saponin and oil, CI = Confidence interval,OD = Optical density, SE = Standard error

Table 6

Mean percentage inhibition of antibody response of cattle against FMDV serotype O as measured by SPCE.
Adjuvantformulation	Percentage of inhibition (PI%)
Adjuvantformulation	Day 0	Day 7	Day 14	Day 21	Day 28	Day 42
Control	51.39	52.78	52.43	59.03	47.51	45.59
AS	43.75	63.19	70.14	76.24	88.29	92.72
AS boost	50	70.14	73.26	82.76	93.49	95.4
Oil	45.48	70.14	81.25	88.12	96.93	98.08
AS + oil	45.14	67.71	81.25	93.87	98.85	98.47
AS = Aluminum hydroxide gel and saponin, AS boost = Aluminum hydroxide gel and saponin boosted, AS + oil = Aluminum hydroxide gel, saponinand oil

Immune response result for FMDV serotype SAT 2

In similar pattern to immune responses recorded for the other two serotypes (Serotype A and Serotype O), the highest mean antibody level and percent of inhibition were observed in groups inoculated with vaccine formulation containing a mixture of aluminum hydroxide gel, saponin and oil, and oil alone (Tables 7 and 8, Figs. 5 and 6). Whereas, the results showed that groups immunized with the vaccine prepared with aluminum hydroxide gel and saponin induced less antibody response and percent of inhibition followed by groups vaccinated with aluminum hydroxide gel and saponin with booster dose.

Table 7

Mean optical density (OD) of antibody response of cattle against FMDV serotype SAT 2

Days post vaccination		Control (mean OD with CI)		AS (mean OD with CI)	AS boost (mean OD with CI)		Oil (mean OD with CI)	AS + oil (mean OD with CI)	P value
Day 0	1.59 (1.21–1.97)		1.30 (0.82–1.78)		1.47 (1.19–1.74)	1.61 (1.35–1.87)		1.59 (1.34–1.85)	0.3123
SE	0.13		0.18		0.10	0.10		0.10
Day 7	1.52 (1.17–1.88)		1.32 (0.94–1.71)		1.12 (0.83–1.41)	0.89 (0.63–1.16)		0.90 (0.69–1.11)	0.0039
SE	0.13		0.15		0.11	0.10		0.08
Day 14	1.25 (0.76–1.73)		0.79 (0.63–0.95)		0.92 (0.73–1.11)	0.59 (0.42–0.76)		0.55 (0.45–0.64)	0.0001
SE	0.17		0.06		0.07	0.06		0.03
Day 21	1.50 (1.0-1.93)		0.63 (0.47–0.79)		0.45 (0.30–0.53)	0.29 (0.16–0.40)		0.11(0.07–0.16)	0.0000
SE	0.15		0.06		0.03	0.04		0.02
Day 28	1.59 (1.23–1.94)		0.32 (0.16–0.48)		0.16 (0.08–0.23)	0.08 (0.05–0.11)		0.04 (0.01–0.07)	0.0000
SE	0.12		0.06		0.02	0.01		0.01
Day 42	1.66 (1.27–2.06)		0.23 (0.14–0.32)		0.16 (0.01–0.31)	0.07 (0.30 − 0.11)		0.02 (0.01–0.03)	0.0000
SE	0.14		0.03		0.05	0.04		0.01
AS = Aluminum hydroxide gel and saponin, AS boost = Aluminum hydroxide gel and saponin boosted, AS + oil = Aluminum hydroxide gel, saponin and oil, CI = Confidence interval, OD = Optical density, SE = Standard error

Table 8

Mean percentage inhibition of antibody response of cattle against FMDV serotype SAT 2 as measured by SPCE.
Adjuvantformulation	Percentage of inhibition (PI%)
Adjuvantformulation	Day 0	Day 7	Day 14	Day 21	Day 28	Day 42
Control	17.19	20.23	34.89	25.37	20.89	17.41
AS	32.29	31.25	58.85	68.66	84.07	88.55
AS boost	23.44	41.67	52.08	77.01	92.04	92.04
Oil	16.15	53.65	69.27	85.57	96.01	96.52
AS + oil	17.19	53.12	71.54	94.53	98	99
AS = Aluminum hydroxide gel and saponin, AS boost = Aluminum hydroxide gel and saponin boosted, AS + oil = Aluminum hydroxide gel, saponin and oil

Challenge test result

The challenge study which involved intradermal injection of experimental cattle on the upper tongue with 10^6.4 TCID50/ ml of homologous FMDV serotype O at 28 days after vaccination did not develop any clinical disease and lesion of FMD. Furthermore, the body temperature of each cattle recorded daily for seven consecutive days did not exhibit any elevation in temperature (remained below 40 ^o C).

Foot-and-Mouth Disease (FMD) is an acute, febrile, and highly contagious vesicular disease of cloven-hoofed animals caused by FMDV. It is devastating disease of livestock causes significant economic losses following an outbreak (28). Control of the disease particularly in endemic areas could be achieved through vaccination with the use of quality vaccine prepared with locally circulated serotypes and effective adjuvants (29). Adjuvant have been used for more than 70 years even when the mode of their action had not been understood (30). The use of adjuvants in inactivated FMD vaccine formulation plays a vital role to improve its performance in provision of rapid and long-lasting protective immunity.Thus, the findings of the present study are imperative in contributing valuable information in the understanding of the effective formulations of FMD vaccines and selection of adjuvants used in the eradication of the disease.

Overall, in all cattle vaccinated with various experimental vaccine formulations SPCE result indicated that there was decrease in OD value of a conjugate antibody during post vaccination period. (Table 3–8 and Fig. 1–6), which is because of the competition between antibodies in test sera and enzyme conjugated antibody for the same antigen. The decrease in OD value is indirectly proportional to antibody level in the test serum. This in turn, indicates increase in antibody titers and percentage of inhibition as antibodies available in test sera impede binding of conjugated antibody (Tables 3, 5 and 7).

Evaluation of antibody responses to the serotype A, O and SAT 2, aluminum hydroxide gel and saponin, oil-based, and combination of aluminum hydroxide gel and saponin with oil-adjuvanted vaccines revealed considerable variation both in the rapidity of development and magnitude of humoral immune response (Fig. 2, 4, and 6).The variation in immune response is related primarily to difference in formulation and booster injection to experimental animals.

In groups vaccinated with aluminum hydroxide gel and saponin formulations in all the three serotypes used in this study, a booster dose has shown a statistically significant difference in mean antibody response compared to groups without a booster dose (p < 0.05). This is in agreement with (31) and (21), who described the poor immune response elicited to inactivated FMD vaccine formulated with aluminum hydroxide gel and saponin necessitate relatively frequent revaccinationsto provide protective immunity. (29) were also suggested that among factors that limit effective performance of inactivated FMD vaccine the requirement for repeat boosting is one factor due to short duration of protection induced by aluminum hydroxide gel and saponin without boosting. Also, similar results obtained by (32) in cattle, suggested that better immune response in cattlecould be effects of booster dose.

The antibody response between aluminum hydroxide gel and saponin boosted and oil-based group were not statistically significant for the three serotypes until day 21 post vaccination (p > 0.05). Asdescribed by (33), this may be due to the reason that oil emulsions adjuvant function as delivery systems by the formation of depot effect that traps antigen at the injection site and by slowing the release of antigen to induce a long-lasting immune response. However, the mean antibody response reached to the maximum in aluminum hydroxide gel and saponin boosted group for serotype A, O and SAT 2 on day 28 and 42 were significantly lower than that of oil group (p < 0.05). These results were concordant with those reported by (34), who found that oil adjuvant elicited a better immune response than did aluminum hydroxide gel and saponin vaccine.

The use of combined formulationof aluminum hydroxide gel, saponinand mineral oil adjuvant is not common. However, in the present study, the mixture of aluminum hydroxide gel, saponin and mineral oil adjuvant for inactivated trivalent foot and mouth disease vaccine was evaluated. The ELISA results and statistical analysis revealed that this group showed relatively a higher post vaccination antibody response than that without mixingagainst the three serotypes of FMDV suggesting that the combination of aluminum hydroxide gel; saponinand mineral oil have a great impact on the immune response. This was partly in agreement with (33), in that FMD vaccine prepared with mixing aluminum hydroxide gel and oil-based adjuvant is considered the best in the humoral immune response than oil alone. In the study by (35) conducted in mice, showed that combinations of aluminum hydroxide gel and oil-based adjuvant revealed the highest protection rate. Moreover, the results also partially corresponded to previous work conducted by (36), who reported a combination of oil with a gel enhanced the immune response in experimental animals and concluded that oil improves the effects gel of adjuvant.

On the other hand, a synergy between oil and saponin for immune response has been reported in some studies. (3), observed a strong antibody response in cattle vaccinated with FMD vaccine formulated with combined oil and saponin adjuvants. Recently, the other study conducted by (37) in guinea pigs and cattle with monovalent FMDV serotype O, also reported that oil-based FMD vaccines containing saponin induced higher antibody level than vaccine without saponin.These findings are partially corroborating with the finding of the present study that enhanced antibody response was obtained by the use of aluminum hydroxide gel and saponin in combination with mineral oil adjuvant in a FMD vaccine compared to other adjuvants.

The groups immunized with combination of aluminum hydroxide gel, saponin and mineral oil induced the higher humoral immune response in comparison to groups vaccinated with aluminum hydroxide gel and saponin. According to this finding, the higher immune response induced could be because of the effects of mineral oil adjuvant added in the formulation. This suggests that oil adjuvant improves the effects of aluminum hydroxide gel and saponin adjuvants.

In this experimental study the virus prepared for challenge test did not develop any typical clinical signs and lesions of FMD in control group. This might be due to the serial passage of the virus used for vaccine production (laboratory adapted virus) which could lead to losing its infectivity. This is in agreement with finding of (21), who found that the control cattle did not develop any clinical disease. As a result, further potency test of the formulated vaccines was not carried out.

This study demonstrated that cattle vaccinated with single injection of vaccine formulations containing aluminum hydroxide gel and saponin as an adjuvant had lower antibody response compared tocattle boosted at day 14 with the same vaccine formulation. These observations confirmed that the booster dose is necessary to enhance the immune response of aluminum hydroxide gel and saponin adjuvanted vaccine against FMD.Besides, trivalent FMD vaccine prepared with oil and mixture of aluminum hydroxide gel, saponin and oil adjuvant vaccine elicited better immune response than the other adjuvant formulation.It could be concluded that oil-based and aluminum hydroxide gel and saponin with oil adjuvanted vaccine could replace the aluminum hydroxide gel and saponin in FMD vaccine. Future experimental research on evaluation of these adjuvants including duration of protection and biological characterization of viral infectivity is highly recommended.

ARERC Animal Research Ethics Review Committee

AS Aluminum hydroxide gel-Saponin

BHK Baby Hamster Kidney

ELISA Enzyme-Linked Immune Sorbent Assay

FAO Food and Agriculture Organization

FMD Foot and Mouth Disease

FMDV Foot and Mouth Disease Virus

NAHDIC National Animal Health Diagnostic and Investigation center

NVI National Veterinary Institute

O/W Oil in Water

OIE Office International des Epizooties

PANVAC Pan African Veterinary Vaccine Center

PBS Phosphate Buffer Saline

SAT Southern African Territory

SPCE Solid Phase Competitive Elisa

TCID Tissue Culture Infectivity Dose

Description of the Study area

The study was conducted from November 2020 to June 2021 in the National Veterinary Institute, Bishoftu, Ethiopia. All experimental works were done at NVI and ELISA assay to measure antibody response against FMDV was carried out at National Animal Health Diagnostic and Investigation center (NAHDIC), Sebeta, Ethiopia.

Experimental animals

The animals used in this experimental study were supplied by National Veterinary Institute. They were local breed male cattle aged three to four years old, apparently healthy or free from any clinical signs. Prior to vaccination, all cattle were ear tagged and identity number was given. All the study animals used for this experimental study were bled and tested for the presence of anti-FMDV nonstructural protein (NSP) antibodies using the FMD nonstructural enzyme-linked immunosorbent assay (NS-ELISA) (IDEXX Switzerland). Only cattle with a negative result with the FMD NS-ELISA were used for the study. They were randomly allocated into five groups with six cattle in four groups (6 cattle/group) and five in control /non-vaccinated groups anda total of twenty-nine (29) cattle were used. Throughout the study period, they were kept separately and cared for at the animal rooms of NVI with adequate provision of feed and water.The cattle in each vaccine group were vaccinated subcutaneously (SC) with inactivated trivalent (serotype A, O and SAT 2) FMD vaccine formulated with different adjuvants in the middle of the cervical area and blood samples were collected at 0, 7, 14, 21, 28 and 42 days after vaccination for serology testing. Generally, these cattle were used in the evaluation of humoral immune response induced because of vaccine formulated with different adjuvants and booster dose.

Cell culture

Cryopreserved suspension of Baby hamster kidney cell line (BHK-21) was provided by Pan African Veterinary Vaccine Center (PANVAC) used for vaccine preparationand virus titration according to method described by (38). The cell preserved in liquid nitrogen (-196 ^OC) were thawed in water bath at 37 ^OC. The thawed cellswere transferred into falcon tube and centrifuged at the speed of 1500 rpm for 10 minutes and the supernatant was discarded. Then about 5 ml of complete Minimum Essential Media (MEM) containing 10% calf serum and Tryptose phosphate broth (TPB) was added to the pellet and transferred to flask of 25 cm². The culture medium and cells were mixed well by pipetting. The flask was allowed to incubate at 37 ^OC and 5% CO₂ in a humidified incubator for 48 hours and the cell growth was followed up to show confluence. After 48 hour of incubation, good cell growth was seen under inverted microscope which was indicated by confluent cell (Fig. 7, left). Once confluent cell was obtained, the medium was decanted, and the flask was washed 3 times with sterile phosphate buffer solution (PBS) to remove the residual of culture medium. Washing the flask with PBS was followed by addition of trypsin and EDTA to detach or dissociate adherent cells from the flask and observed for 3 minutes. After the cell confirmed to be detached, the cell was re-suspended in complete MEM media following by trypsinization process to get a single cell. Then the cell was passaged in flasks of 75 cm² and sub-cultured into another flask of 75 cm² accordingly.

Vaccine preparation

The FMDV serotype A/21/ETH/2008, O/ETH/38/2005, SAT 2/ETH/2009 locally isolated antigenwas used for vaccine preparation using BHK-21 cell line. Maintenance media (Hanks media) was also used for cultivation and inoculum preparation as described by (39). Before inoculation of the virus into the grown cell, the inoculum was prepared by diluting FMDV in1:100 dilutions in Hank’s media and antibiotic were added in the inoculum.For sterility test, little amount of inoculum was inoculated into bacteriological media to identify the presence of any type of bacteria. The old media was removed. Consequently, the confluent BHK-21 monolayer cell was allowed to be infected with the diluted viruses on each flask for the three serotypes aseptically. The flasks were incubated at 37 ^OC and followed for 17–24 hours for development of cytopathic effect (CPE) by the virus. Within 24 hours of post infection, complete development of CPE in BHK-21 cell culture were observed under inverted microscope. CPE was indicated by the rounding and flattening of the cells and finally death of the cell that indicates the presence of the virus (Fig. 7, right). The culture supernatants of virus adapted and grown on BHK-21 cell monolayers were separately harvested after 24 hours post-infection and poured into sterile container. The sterility test was conducted on bacteriological media. Then, the virus supernatant was preserved aseptically at -20^OC until further work.

Purification and inactivation of the virus

The harvested FMDV serotype A, O and SAT 2 from infected BHK-21 cell culture were initially concentrated by addition of 50% of ammonium sulfate and stirred at low speed overnight (40). Therefore, the viruses were further purified by using high-speed centrifugation at 3000 rpm for 30 minutes to remove unwanted substances or cell debris according to described by (41). The concentrated FMDV serotype A, O and SAT 2 were re-suspended in PBS with pH of 7.2. Therefore, they were pooled in 1:1:1 ratio and inactivation of the viruses were achieved by using a final concentration of 0.06% of formaldehyde according to method described by (42). Complete inactivation of the virus was confirmed by using BHK-21 cell culture. Finally, purified and inactivated FMDV serotypes were stored at + 4^o C until used.

Adjuvant preparation

Mineral oil adjuvant

Water in oil (W/O) emulsion or incomplete Freund's adjuvant (IFA) adjuvant used in this experimental study was prepared according to NVI protocol by mixing the following mineral oils: 3.6 % Montanide 888, 5 % Tween 80, and 48 % PBS and 43.3 % Marcol 52. The mixture was homogenized and sterilized by autoclaving at 121^oC for 15 minutes. The prepared adjuvant was stored at room temperature in dark place until used.

Saponin

The saponin adjuvant was prepared in 10 %, filtered by 0.22 µm pore filter sheet for sterilization. Final concentration of 0.3 % v/v was used to formulate with the vaccine (NVI protocol).

Aluminum hydroxide gel

10 % of aluminum hydroxide gel was prepared according to method described by (43) and autoclaved at 121 ^oC for 15 minutes and stored at 4 ºC until used.

Vaccine formulation with adjuvants

Formulation of the vaccine with adjuvants were performed as follows: Formulation 1: inactivated trivalent FMD vaccine with aluminum hydroxide geland saponin (AS), Formulation 2: inactivated trivalent FMD vaccine mixed with mineral oil adjuvant, Formulation 3: inactivated trivalent FMD vaccine with combination of AS and mineral oil adjuvant. In these formulations, 0.3% of saponin and 10% of aluminum hydroxide gel were used to formulate with the vaccine and oil-based adjuvant was formulated with aqueous vaccine in ratio of 50:50 according to (43).

Experimental design

Longitudinal study was applied to evaluate immune response of the experimental animals. Twenty-nine (29) male cattle were grouped into five groups as follows to evaluate the immune response elicited by different formulation of adjuvants as well as to evaluate effect of booster dose. The first group was vaccinated with inactivated trivalent FMD vaccine adjuvanted with the conventional aluminum hydroxide gel and saponin (AS) (without boosting). The second group was vaccinated similarly as first group with AS adjuvanted vaccine and booster dose was given for this group at 14 days post vaccination. The third group was vaccinated with mineral oil adjuvanted inactivated trivalent FMD vaccine. The fourth group was vaccinated with vaccine formulated with AS and oil and the last group was left as control/non-vaccinated. In this experimental study, every vaccine formulates with a different adjuvant were equally likely applied to each group and every experimental calf within the group. Allocation of adjuvants to each calfwas on a random basis to distribute experimental error equally for all groups. At the end of the experiment humoral immune response of each group were measured by using solid-phase competitive ELISA (SPCE) and the result of antibody response of each calf within each group was recorded.

Challenge with live virus

After twenty-eight days of post vaccinationthe vaccinated and control group were challenged by intradermal inoculation in the upper surface of the tongue with 0.1 ml of the virus per site according to the protocol described by (44, 27). All the challenge experiments were carried out in a highly contained environment at NVI. Locally isolated live FMDV serotype O provided by NVI and having 10^6.4TCID₅₀ /ml infectivity titer was used as a challenge virus. After challenge the cattle were restrained and carefully examined daily for any clinical signs of FMD and lesion, inside the mouth in the muzzle, on the tongue and feet for two weeks. Additionally, their rectal temperatures were monitored twice per day for the first seven consecutive days and recorded.

Immunization of the animals

A total of 29 local zebu breed male cattle, aged 3 to 4 years’ old, held at an experimental room of animal facility and quarantine department if NVI were selected for this study. The cattlewere randomly grouped into five groups and the cattle in each group were vaccinated subcutaneously (SC) using individual syringes of 10ml and 21G needle for each group in the middle of the cervical area with 4 ml of one of the following vaccines. Group 1 were vaccinated with trivalent FMD vaccine adjuvanted with conventional AS and group 2 were similarly vaccinated as group 1, but they took booster dose on 14^th day post vaccination. Group 3 were vaccinated with vaccine formulated with oil-based adjuvant. Group 4 were vaccinated with vaccine containing combination of standard AS and oil. The fifth group were left as control. After the cattle given first dose with 4 ml SC (at 0 day), all the group except group 2 were immunized twice at the interval of 14 days post vaccination with the same protocol and dose used for primary vaccination to boost the first immunization.

Blood and serum sample collectionand processing

The blood sample were collected from each group of animals for detecting humoral immune response of vaccinated and non-vaccinated cattle on day 0, 7, 14, 21, 28, and 42 days post vaccination. About 5 ml blood sample was collected from all experimentally immunized and control group in a clean sterile plain vacutainer tube from the jugular vein using individual syringe and vacutainer needle of 0.8*38 mm for each animal. Prior to collection the animals were restrained in crash and site of collection was palpated to determine the location of the jugular vein located in the lower neck and disinfected with 70 % ethanol. The vein was pressed firmly by thumb and the needle was inserted into engorged vein by free hand and the blood was collected in plain vacutainer tube. The collected blood was left at room temperature for 24 hours to clot. After 24 hours, sera were harvested aseptically into clean cryovial tubes which were labeled as day 0, 7, 14, 21, 28, and 42 as well as by a tag number of the animals and stored at -20 ^oC until testing. Then, the serum samples were labeled and transported to NAHDIC with the ice box for ELISA assay.

Serological assays

Solid phase competitive ELISA (SPCE)

Serum samples collected from the vaccinated and non-vaccinated cattle were tested for the antibody response against the three serotypes of FMDV (A, O and SAT2). The testwas carried out by using solid phase competitive ELISA (SPCE) assay for detection of structural proteins specific to the three FMDV serotypes (A, O, and SAT 2). The assay is applied to measure antibodies in serum samples of FMDV infected or vaccinated animals using a selected neutralizing anti-FMDV monoclonal antibodies (mAbs), specific for FMDV serotypes. In SPCE assay the test sera were incubated with the trapped inactivated antigen, enabling the specific antibodies present in the sample to bind to the respective antigen. Then, the ant-FMDV mAb, conjugated with peroxidase is dispensed. The reaction of conjugated antibody with homologous antigen were inhibited by antibodies of positive sample previously bound to the virus, while in the case of negative sample the conjugated mAb were bound to the virus and the reaction is appreciated by color development (45).

Therefore, in the present study, the SPCE kits ready to use were used to measure antibody levels elicited by FMD vaccines containing serotype A, O and SAT 2, the test was performed aseptically for each serotype according to the instructions given in the manual (IZSLER Brescia Italy). 96-wells ELISA microplates supplied pre-coated with inactivated antigen of FMDV serotypes captured by the homologous mAb were used. In the assay, 3 plates/sample coated with specific antigens of type A, O and SAT 2were used. The first two wells A1 and B1 were used for positive control (i.e., 1/10 and 1/30 dilution of positive sera were added in A1 and B1 wells respectively). In the A1 the positive sera (7.5μl) and ELISA buffer (67.5μl) were mixed using monochannel micropipette and in B1, 50μl buffer was dispensed. After mixing, 25μl of the positive control serum was transferred from the dilution of 1/10 to the subsequent well B1 to obtain the dilution of 1/30 and mixed. Then, without changing the tip, 25μl was pipetted from well B1 and discarded to maintain the final volume of 50μl /well. Accordingly, 50μl negative control was distributed in each of the four wells in positions of C1, D1, E1 and F1. The test was continued with distribution of 45μl/well ELISA diluent buffer and 5μl/well of test sera to gain the final dilution of 1/10 and mixed using multichannel pipette. The plates were covered and incubated at room temperature for 60 minutesto allow antigen-antibody (Ag-Ab) reaction. After incubation, 25μl/well HRPO (Horse Reddish Peroxidase)-conjugate diluted at 1/10 dilution was added to each well. Again, the plates were covered and incubated at room temperature for 60 minutes. After 1-hour incubation, the fluid was removed from the wells. The plates were filled with 200μl/well washing solution containing PBS-tween20 diluted in distilled water (1/10 dilution) and left at room temperature for 3 minutes without removing the solution. Washing solution was removed and washing was repeated 3 times. The last washing was left at room temperature for 5 minutes (in total of four washing cycles). Therefore, the plates were tapped firmly onto clean absorbent towel to remove residual fluids. 50 μl/well substrate/chromogen solution was added to all wells and the plates were allowed to incubate at room temperature for 20 minutes in the dark place. Finally, stop solution (50μl/well) was added to stop the reaction between substrate and enzyme and the plates were gently shaken prior to reading. Immediately after addition of stop solution, optical density (OD) of each well was measured at wavelength of 450 nm using microplate reader. The ELISA plate absorbance was converted to the percent inhibition (PI) value. The PI 70% or above at 1/10 dilution, was considered the cutoff and the cattle were regarded as antibody positive.

A percentage of inhibition (PI) was calculated according to the formula described by (kit manufacturer and OIE,2018): (100 – [mean optical density of the group/mean optical density of the negative control] × 100). The percentage of inhibition implies the competition between antibody present in test sample and HRP conjugated guinea-pig anti-FMDV antisera for the specific FMDV antigen pre-coated on the ELISA plate (27). Therefore, PI is directly proportional to antibody level in the test sera.

Criteria for test validity

The spectrophotometric reading of the negative control wells is expected to give OD value of 1 or higher for FMDV serotype SAT2 and O, and 0.8 OD or more reading in negative control wells for FMDV serotype A. While, in wells of the positive control serum expected to give 90 % or higher inhibition at 1/10 dilution and >50% at 1/30 dilution for serotype A, and O, 80% or higher at 1/10 dilution and >50% at 1/30 dilution for SAT2 serotype (Table 9).

Table 9: Criteria of test validity for the three FMDV serotypes

FMDV serotypes	Expected ODin negative control	Expected PI % at 1/10 dilution	Expected PI % at 1/30 dilution
A	≥0.8	≥90	>50
O	≥1	≥90	>50
SAT 2	≥1	≥80	>80

Virus titration

The three FMDV serotypes used as the vaccine strains were titrated by using 2% incomplete MEM added to 96-microtiter plate wells. Tenfold serial dilution starting with 10⁻¹ by mixing 0.5 ml of virus in 4.5 ml of MEM. Similarly, consequent transfer of 0.5 ml from the first virus dilution to the next using aseptic and sterile pipette for each dilution and finally 0.5 ml was discarded without changing the pipette. Thus, from each virus dilution in the universal bottles (10^-1 to10^-8),100 µl /wells were dispensed into the wells of their respective rows on 96-wells micro titer plates filled with 100µl/wells of BHK-21 cells which was prepared 48 hours prior to infection. The plates were sealed and incubated at 37 °C in an atmosphere of 5 %Co₂ incubator for 48 hours. The cytopathic effect (CPE) was observed under a microscope after48 hours and the infectivity titer of each virus were determined according to Spearman–Karber method (47). The titer of the virus was expressed as log₁₀ TCID₅₀ /ml.

Sterility and safety testing of prepared vaccine

The prepared vaccine was tested for the presence of aerobic and anaerobic bacteria, fungal and mycoplasma contaminants by culturing of vaccine sample in thioglycolate broth, Sabouraud agar and mycoplasma medium. Complete inactivation of the virus was also tested for safety in vitro on BHK-21 cell line (48).

Data management and analysis

Data from SPCE ELISA result was recorded on Microsoft Excel spreadsheet. Therefore, data analysis was performed using analytical software STATA version 12. Analysis of variance (ANOVA) was used to determine statistical significance between the adjuvant formulation with respect to their immune response. Students t. test was also used to compare pairwise comparisons of mean of adjuvants. p<5% (0.05) at 95 % confidence interval (CI) was used to report the effects as statistically significant.

Ethical approval

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. In line with this all the experimental protocols were approved by Addis Ababa University, College of Veterinary Medicine and Agriculture (AAU-CVMA), Animal Research Ethics and Review committee (Ref No: VM/ERC/27/06/13/2021). The authors confirm that all methods are carried out and reported in accordance with Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines. The authors also confirm that all methods are carried out and reported in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request and with permission of IRB and/or other responsible bodies in Ethiopia.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was financially supported by the National Veterinary Institute (NVI), Bishoftu Ethiopia. The funding organization had no any role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript

Acknowledgments

The authors would like to thank the National Animal Health Diagnostic and Investigation Centre (NAHDIC) for supporting the study.

Author Contributions

GA conducted the experimental work. GA, BG, EG, BME conceived the idea and designed the study. BME and GA wrote the draft of the manuscript and completed the final version for submission. BME supervised the work. GA analysed and interpreted the data. BB, TA supported the experimental work. AM supported the lab work and data curation. FD participated the in the development of the idea, co-supervised the work and edited the final version of the work before submission

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No competing interests reported.

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Evaluation of different adjuvant formulations of trivalent Foot and Mouth Disease vaccine in cattle

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