Swiss albino mice, 21 - 30 days old were used in the potency test and measuring vaccines’ validity kept for different periods post preparation at 4˚C.

Twenty suckling albino mice 3 - 5 day old were used to confirm complete inactivation of RVFV. The Laboratory Animal Breeding Unit of the Veterinary Serum and Vaccine Research Institute (VSVRI), located in Abbasia, Cairo, provided them.

Eight new born healthy native breed lambs 7 - 10 days old were used in order to evaluate the manufactured RVF vaccine’s safety. The Laboratory Animal Breeding Unit, Veterinary Serum and Vaccine Research Institute (VSVRI), Abbasia, Cairo, provided them.

Fifteen adult native breed sheep were tested with SNT and determined to be free of RVF antibodies, after which they are kept in bug proof stables with a balanced diet and enough water under rigorous sanitary conditions and used for evaluation of humeral and cell mediated immune response induced by the prepared vaccines.

Rift Valley Fever Virus (RVFV) (ZH501) with a titer of 10⁷ TCID₅₀/ml was kindly supplied by Rift Valley Vaccine Research Department (RVFVRD), VSVRI and used for vaccine preparation and serological testing.

RVFVRD provided baby hamster kidney cells (BHK21) monolayer, which were employed for RVF propagation, titration, and SNT-based assessment of the humeral immune response.

Lubrizol Co. provided it as a powder, which was then mixed in hot water to create stock solution that was 1% aqueous [20] . After autoclaving the prepared solutions for 20 minutes at 121˚C to heat sterilize them, it was kept at 4˚C until it was needed again.

It was purchased as Lot No. 11-274-30 from Alliance Bio Company, USA, and added to the produced local vaccination at a 20% concentration [21] .

RVF virus (ZH 501) was propagated under biosafety measures in BHK cells for three successive passages to increase the virus yield.

According to [22] , the RVF virus was titrated in BHK21 cell culture using the microtiter technique, and [23] calculated the viral titer as log¹⁰ TCID₅₀/ml.

By adding 1% Binary Ethylenamine (BEI) in 0.2 NaOH to the viral solution, the RVF virus was rendered inactive, resulting in a final concentration of 0.001 M of BEI. After thoroughly mixing the virus and BEI combination, sodium bicarbonate was added to bring the pH down to 8.0. For 24 hours, the virus was kept in an incubator at 37˚C in a magnetic stirrer as described [24] .

1) In cell culture: According to [25] , inactivated RVF viral samples were examined for the presence of a virulant virus in tissue culture by introducing the inactivated virus into a BHK confluent monolayer, which allowed for daily microscopic inspection for detection of cytopathic effect (CPE).

2) In baby mice: By inoculation of baby mice (3 - 5 days old) with 0.03 ml of inactivated virus as said by [26] and [27] then mice were kept for 10 days with daily examination. Mice that died in the first 24 hours were thrown out.

The two prepared vaccine formulae were tested for sterility on specific media to prove their freedom of aerobic and anaerobic bacteria; fungi and mycoplasma following up the directions of [28] .

Three lambs received 10 ml of the prepared vaccine formula-1 (5 ml by S/C and 5 ml by I/P) and another three lambs received the same dose of the prepared vaccine formula-2 though the same routes while the remained two lambs were kept without vaccination as a control. For a period of two weeks, a daily clinical observation was conducted to record any increase in body temperature or abnormal clinical findings.

Additionally, for seven days after vaccination, vaccinated sheep were monitored clinically every day, with body temperature and any unusual clinical indications related to the immunization being recorded.

1) In mice (detection of ED₅₀): Five-fold dilutions of each vaccine formula, ranging from 1:1 to 1:625 in appropriate medium, were prepared in accordance with [29] . Subsequently, a week apart, two doses of 0.2 ml of the tested vaccine were administered intraperitoeally (I/P) to each mouse in each of five groups of weaned mice (10 mice/group). Vaccinated mice were challenged with 0.1 ml RVF challenge virus (10³ MIPLD₅₀/ mouse inoculate) inoculated I/P 7 days after the second dosage. In addition, there were two additional groups of mice: unvaccinated and not challenged, as control negative group and another unvaccinated group challenged with RVF virus as a control positive. For duration of 21 days, all mouse groups were housed under close observation. The ED50 was calculated according to [23] .

2) In Sheep:

a. Scheme of sheep vaccination: Following the planned experimental work, 15 sheep were used to test the two prepared vaccination formulae. They were split up into three groups of five sheep each, kept in insect-proof stables, fed a balanced diet, given enough water, and subjected to stringent cleaning requirements. Using a dosage of 1 ml of each formula/animal (inoculated S/C) given in two doses with one month interval. The first group received the aluminum hydroxide gel inactivated RVF vaccine (formula-1), while the second group received the carbopol inactivated RVF vaccine (formula-2). The third group was kept as control without vaccination.

b. Blood samples: They were collected at 0 day before vaccination, and on day intervals post vaccination (1, 3, 5, 7, 10, 14, 21 and 28 days) then monthly till the end of experiment (12 months).

c. Serum samples: They were obtained from all sheep groups for determination of humeral immune response and detecting the level of Interferon and Interleukin.

d. Evaluation of humeral immune response:

Cut off = mean of negative control samples + 3 Standard deviation of mean negative control samples (SD)

e. Evaluation of cell mediated immune response:

In order to determine the amount of sheep interferon gamma and interleukin-2 in the samples under investigation, the evaluation of interferon gamma and interleukin-2 (IL-2) in sheep serum was conducted using a double antibody sandwich ELISA kit (Double Antibody Sandwich Enzyme linked Immunosorbent Assay, Catalog No. 201-07-0063, MABTECH ® Sweden). After adding the stopping solution, the optical density was measured within 15 minutes at a wavelength of 450 nm using specterophotometer.

The shelf life of Carbopol adjuvanted inactivated RVF vaccine was determined by application of potency test till the 16^th month in comparison with ALOH adjuvanted inactivated RVF vaccine as the permissible limit the ED₅₀ is (0.02) according to [32] and [33] .

All results were analyzed via the SPSS Inc. software version 26 (IBM Corp., Armonk, NY, USA). The normality among the experimental groups was determined utilizing Shapiro-Wilk’s test. Levene’s test was used for testing the homogeneity of variance. One-way ANOVA and Tukey’s tests were employed to evaluate the significant variations between the mean value that were normally distributed and had homogeneity of variance. When the p-value was lower than 0.05, statistically significant variations were considered. All graphs were generated via the GraphPad Prism software Version 8 (San Diego, USA).

In cell culture, the RVF virus (ZH501) was propagated for three consecutive passages. From the first passage (10⁷ TCID50/ml) to the third passage (10^7.8 TCID50/ml), the viral titer was shown to have grown progressively, as indicated in Table 1 . The obtained titer at the third passage was used to prepare the experimental vaccine formulae.

During the 10-day observation period, infected newborn mice did not exhibit any aberrant post-inoculation indications or fatalities, and as Table 2 illustrates, injected BHK cell culture did not display any CPE.

*Sterility test: The prepared vaccines were cultivated on several specialized medium to identify the development of bacteria, fungi, and mycoplasma. Both formulae were determined to be free from mycoplasma, fungus, and aerobic and anaerobic bacteria.

*Safety test: The test showed that, during the course of ten days of monitoring, the lambs that had received the vaccination neither exhibit any aberrant indications or post-vaccinal responses, nor show body temperature increase ( Table 3 ).

*Potency test of the prepared RVF vaccine formulae:

*In mice: Determination of the ED₅₀ showed that formula-2 (adjuvanted with Carbopol) had 0.0012 ED₅₀/ml while formula-1 (adjuvanted aluminum hydroxide gel) had 0.0019 ED₅₀/ml showing their potency whereas the permissible limit is less than 0.02/ml.

Gp1: Group-1 vaccinated with formula-1 (with ALOH gel adjuvant); Gp2: Group-2 vaccinated with formula-2 (with Carbopol adjuvant); Gp3: Unvaccinated control group.

*In sheep (humeral and cell mediated immune response):

According to the findings in Table 4 , the neutralizing antibody indices in groups 1 and 2 significantly increased in value, reaching the protective level in the first week for Gp. 2 which received the Formula 2 vaccination and the second week for Gp. 1 which received the Formula 1 vaccination (1.5, 1.2, respectively), and maintained their protective levels until 40 weeks after immunization in GP.1 and 48 weeks after vaccination in GP.2 (1.5, 1.6), respectively. These results mirror those of the ELISA test, which are shown in Table 5 .

Gp1: sheep vaccinated with ALOH Gel inactivated RVF vaccine, Gp2: sheep vaccinated with Carbopol Adjuvanted inactivated RVF vaccine, Gp3: unvaccinated control, WPV: weeks post-vaccination. Results are expressed as mean ± standard error of the mean (SEM). a-c Means with different superscript letters indicate significant differences at p < 0.05. The protective index is 1.5.

Gp1: sheep vaccinated with ALOH Gel inactivated RVF vaccine, Gp2: sheep vaccinated with Carbopol Adjuvanted inactivated RVF vaccine, Gp3: unvaccinated control, WPV: weeks post-vaccination. Results are expressed as mean ± standard error of the mean (SEM). a-c Means with different superscript letters indicate significant differences at p < 0.05. Cut off: 0.250.

Figure 1 shows the neutralizing indices in vaccinated and control sheep groups. There were significant differences in neutralizing indices among different experimental groups (p < 0.0001). Tukey’s post-hoc test showed that sheep in the Gp2 group showed the most significant increase in neutralizing indices when compared with the unvaccinated control group in all time points except 12WPV. At 12WPV, sheep in Gp2, and Gp1 groups showed the most significant increase in the neutralizing indices (3.4, and 3.2, respectively) when compared with the unvaccinated control (Gp3) group (0.5), with no significant differences between Gp1, and Gp2 groups ( Table 1 and Figure 1 ).

Figure 2 displays the optical densities at different periods post-vaccination in vaccinated and control sheep groups. There were significant differences in optical densities among different experimental groups (p < 0.001) at different time sampling (1WPV-52WPV). Tukey’s post-hoc test showed that Sheep in Gp2 and Gp1 showed the most significant increase in the optical densities when compared with the unvaccinated control (Gp3) group.

As can be seen in Table 6 , the serum level of IL-2 in sheep groups 1 and 2 increased significantly from the first day after vaccination, but this increase was more pronounced in group 2 that received the Carbopol adjuvanted inactivated RVF vaccine. Groups 1 and 2 reached their peak levels at 7^th (0.810) and 10^th (1.99) days after vaccination, respectively. These levels steadily decreased until the experiment’s end.

Gp1: sheep vaccinated with ALOH Gel inactivated RVF vaccine, Gp2: sheep vaccinated with Carbopol Adjuvanted inactivated RVF vaccine, Gp3: unvaccinated control, DPV: days post-vaccination. Results are expressed as mean ± standard error of the mean (SEM). a-c Means with different superscript letters indicate significant differences at p < 0.05.

Figure 3 displays the concentration level of serum IL-2 at different periods post-vaccination in vaccinated and control sheep groups. There were significant differences in the concentration level of serum IL-2 among different experimental groups (p < 0.001). Tukey’s post-hoc test showed that sheep in the Gp2 group showed the most significant increase in the concentration level of serum IL-2.

The results illustrated in Table 7 revealed that the serum level of IFN-γ in sheep groups (0.150, 0,235) increased significantly from the first day after vaccination, but it was more noticeable in group 2 (vaccinated with Carbopol adjuvanted inactivated RVF vaccine) (0.235). It peaked in groups 1 and 2 at 7^th and 10^th days after vaccination, respectively (0.818, 1.57). These levels steadily decreased until the experiment’s end.

Figure 4 shows the concentration levels of serum IFN-γ at different periods post-vaccination in vaccinated and control sheep groups. There were significant differences in the concentration level of serum IFN-γ among different experimental groups (p < 0.001). Tukey’s post-hoc test showed that Sheep in the Gp2

Gp1: sheep vaccinated with ALOH Gel inactivated RVF vaccine, Gp2: sheep vaccinated with Carbopol Adjuvanted inactivated RVF vaccine, Gp3: unvaccinated control, DPV: days post-vaccination. Results are expressed as mean ± standard error of the mean (SEM). a-c Means with different superscript letters indicate significant differences at p < 0.05.

group showed the most significant increase in the concentration level of serum IFN-γ.

Monitoring of the validity of the prepared inactivated RVF vaccine formulae:

Estimation of the ED₅₀ in response to the permissible limit (0.02 ED50/ml) showed that the validity of ALOH gel adjuvanted inactivated RVF vaccine persisted up to 15 months at 4˚C while that of the Carbopol adjuvanted vaccine still within the permissible limit up to 16 months as shown in Table 8 .

Formula-1: Adjuvanted with ALOH gel, Formula-2: Adjuvanted with Carbopol *MK: Month of keeping; N.B.: The permissible limit is 0.02.