The Development of Dual Vaccines against Lumpy Skin Disease (LSD) and Bovine Ephemeral Fever (BEF)

Dual vaccines (n = 6) against both lumpy skin disease (LSD) and bovine ephemeral fever (BEF) were constructed, based on the BEFV glycoprotein (G) gene, with or without the BEFV matrix (M) protein gene, inserted into one of two different LSDV backbones, nLSDV∆SOD-UCT or nLSDVSODis-UCT. The inserted gene cassettes were confirmed by PCR; and BEFV protein was shown to be expressed by immunofluorescence. The candidate dual vaccines were initially tested in a rabbit model; neutralization assays using the South African BEFV vaccine (B-Phemeral) strain showed an African consensus G protein gene (Gb) to give superior neutralization compared to the Australian (Ga) gene. The two LSDV backbones expressing both Gb and M BEFV genes were tested in cattle and shown to elicit neutralizing responses to LSDV as well as BEFV after two inoculations 4 weeks apart. The vaccines were safe in cattle and all vaccinated animals were protected against virulent LSDV challenge, unlike a group of control naïve animals, which developed clinical LSD. Both neutralizing and T cell responses to LSDV were stimulated upon challenge. After two inoculations, all vaccinated animals produced BEFV neutralizing antibodies ≥ 1/20, which is considered protective for BEF.


Introduction
Lumpy skin disease (LSD) and bovine ephemeral fever (BEF) are two cattle diseases of economic importance with low mortality but high morbidity rates [1][2][3]. LSD is classified as a notifiable disease by the World Organization for Animal Health (OIE); it is characterized by fever, clinical lesions which affect animal hides, reduced milk yields and abortion in pregnant ewes [2,4]. It was initially confined to Africa, but spread to Egypt in 1988, Israel in 1989, followed by the Middle East in the 1990s [5]. More recently, it has spread to Europe and Asia [3,4,6,7] BEF is caused by an RNA virus, belonging to the family Rhabdoviridae, genus Ephemerovirus, group Lyssavirus. Clinically, it presents as a transient disease of 3-4 days, causing sudden fever, salivation, nasal discharge, stiffness and can cause reduced milk production as well as abortion in pregnant ewes and infertility in bulls [1,8]. The disease is endemic in Africa, the Middle East, Asia and Australia [9][10][11]. A live attenuated vaccine, B-phemeral B-phemeral virus administered alone (days 0, 3,10,17,29,38,55,58,65), followed by nine inoculations of BEFV complexed with naked bacteria, six weeks later (days 0, 4, 7, 15, 18, 22, 28, 32 and 35) [39]. The final bleed was taken on day 42 of the second set of inoculations.
Immunofluorescence was used to detect BEFV protein in cells infected with the six recombinants, LSDV(∆SOD)BEFV-Ga, LSDV(SODis)BEFV-Ga, LSDV(∆SOD)BEFV-Gb, LSDV(SODis)BEFV-Gb, LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M. As controls, cells were infected with nLSDV∆SOD-UCT or nLSDVSODis-UCT (negative controls) or B-phemeral (positive control). MDBK cells, seeded in chamber slides, were infected at an MOI of 0.1 for 48 h. The cells were washed and fixed with 4% paraformaldehyde for 10 min, followed by methanol for 30 s before being washed twice with PBS for 10 s. The slides were incubated overnight with rabbit serum (diluted 1:1000), which had been pre-adsorbed with the lysed cellular debris of approximately 4 × 10 7 MDBK cells, which were freeze/thawed and centrifuged at 15,000× g for 10 min. The rabbit serum was added to the cellular debris in 10 mL PBS with 2% BSA and pre-adsorbed for 4 h, after which the cell debris was removed by centrifugation at 3000× g for 7 min and the supernatant was used. After incubation with primary antibody the cells were washed twice with PBS and the secondary antibody was added. For detection of BEFV protein from recombinants expressing Ga, cells were treated with donkey anti-rabbit Alexa488 (green) secondary antibody (Sigma) (diluted 1:500); for detection of BEFV expression from recombinants expressing Gb and Gb-M, cells were treated with donkey anti-rabbit CY3 (red) secondary antibody (Sigma) (diluted 1:500). Cells were incubated with secondary antibody for 1.5 h, washed with PBS (2 × 10 min) and stained with Hoechst solution (1 uL Hoechst in 5 mL PBS) for 1 min. After two washes with PBS (10 min each), the top of the chamber slide was removed and the slide allowed to air dry for 5 min. A drop of mowiol with n-propylgallate (anti-fade) was added and a coverslip placed over the cells. The slides were viewed the following day using an inverted Zeiss LMS 880 with airyscan confocal microscope (Zeiss, Oberkochen, Germany).

Rabbit Immunization
All candidate LSDV-BEFV vaccines were tested in female New Zealand white rabbits of approximately 2 months old, weighing > 2 kg each, with five animals per group. One rabbit died during the acclimatization period and so the control group (inoculated with nLSDV∆SOD-UCT) had only four rabbits. The vaccines were administered intramuscularly (i.m.) as two inoculations of 500 uL into each hind leg. Each animal received three homologous doses of 10 6 TCID 50 given at four-week intervals. Two weeks after the final inoculation blood was collected by cardiac puncture. Serum was heat-inactivated at 56 • C for 45 min.
The rabbit experiments were performed at Stellenbosch University in an insect-free facility and animals were handled by an experienced veterinary surgeon and animal technicians. Approval to perform these experiments was granted by the animal ethics committee at the University of Cape Town, FHS reference number 018_039 and South African Department of Agriculture, Forestry and Fisheries reference number 12/11/1/7/1.

Cattle Immunization and Challenge with Virulent LSDV
The two candidate vaccines LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M were tested in Friesian cattle at the ARC-Onderstepoort Veterinary Research Institute (Transboundary Animal Diseases) facility. Permission was granted to do this experiment by the South African Department of Agriculture, Land Reform and Rural Development (DALRRD), reference number 12/11/1/1. Two groups (n = 10) of cattle > 6 months of age, shown to be LSDV negative by the serum neutralization test (SNT), were vaccinated subcutaneously with LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M, respectively, with an initial inoculation (day 0) of 10 5 TCID 50 per animal followed by a homologous boost of 5 × 10 4 TCID 50 on day 32. Serum was prepared from blood samples collected at On day 201 (169 dpb), cattle were challenged with a total of 1 × 10 7 TCID 50 virulent LSDV (LSDV/Cradock-EC/RSA/1958) per animal, administered intradermally at multiple sites (to mimic vector biting) and intravenously in a 2 mL volume in the neck vein. A control group of three naïve animals, shown to be LSDV seronegative by SNT, were also inoculated in the same way. All animals were monitored for 28 days post challenge and rectal temperatures were recorded. Blood was taken on days 0, 7, 14, 21 and 28 days post challenge (dpc) for whole blood T cell assays as well as neutralization assays (LSDV and BEFV) and BEFV Enzyme-Linked Immunosorbent Assay (ELISA).

LSDV Neutralization
Serum samples were sent to the Diagnostics Services Programme laboratory of the ARC-Onderstepoort Veterinary Institute for lumpy skin disease serum neutralization (LDV-SNT) testing. A neutralization titre greater than or equal to 1/4 was considered positive.

BEFV Neutralization
An in-house BEFV neutralization test was set up at the University of Cape Town to test the ability of serum to neutralize infection of BHK-21 cells with BEFV (B-phemeral vaccine (OBP)). Two-fold serial dilutions were made of sera, starting with 1/5, in DMEM (Gibco, Waltham, MA, USA) supplemented with 1% penicillin-streptomycin (Gibco, Waltham, MA, USA). BEFV was diluted in 1 × PBS to obtain 50 pfu/50 µL (rabbit experiments) or 25 pfu/50 µL (cattle experiment). To each well of a 96-well tissue culture plate, 50 µL of each serial dilution of serum and 50 µL of the diluted BEFV were added. Each serum dilution was tested in ten replicates for the sera from the vaccinated animals and in eight replicates for the pre-immune sera. Following a 2-h incubation at 37 • C in a CO 2 incubator, 100 µL of BHK-21 cells at a concentration of 2 × 10 5 cells/well were seeded and plates were further incubated at 37 • C for 4 days. Wells containing BHK-21 cells only and wells with BEFV + BHK-21 cells were included in each plate as a negative control and virus control, respectively. For the testing of the cattle serum, a rabbit serum sample was selected as an internal positive control. Neutralization titres were determined as the reciprocal of the highest dilution of serum showing inhibition of BEFV infection in ≥50% of BEFV-infected wells [40]. Final SNTs were taken from experiments which showed reproducibility of the positive control. A neutralization titre greater than or equal to 1/5 was considered positive.

BEFV Enzyme-Linked Immunosorbent Assay (ELISA)
The presence of binding antibodies to BEFV was tested using a BEFV antibody ELISA kit (Unibiotest, Wuhan, China). Pre-immune sera and sera from 14 dpb and 30 dpb were diluted 1:10 and added to each well of the 96-well microtiter plates provided, which had been coated with BEFV G protein antigen derived from a truncated Asian sequence. Following 30-min to 1-h incubation at 37 • C, the plates were washed thrice with 1 × PBS containing 0.1% Tween 20 (PBS-T), and 100 µL of rabbit-anti-bovine IgG antibody conjugated with horseradish peroxidase (HRP) were added to each well. The plates were incubated for 30 min to 1 h at 37 • C. After the plates were washed thrice with PBS-T as described previously, 100 µL of 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate was added to each well, and the plates were incubated in dark for 10 to 15 min at 37 • C. The enzymatic reaction was terminated by the addition of 100 µL stop solution to each well. Absorbance was measured at 450 nm (OD 450 ) using a Versa Max Microplate reader and SoftMax Pro Software version 6.3 (Molecular Devices, San Jose, CA, USA). Positive and negative controls were included in the ELISA kit and each sample was tested in duplicate or triplicate. Sera with OD 450 ≤ 0.22 were regarded as negative and OD 450 > 0.30 as positive.

T Cell Assays
Samples were collected pre-challenge and 7, 14 and 21 days post challenge (dpc). Whole blood was mixed 1:1 with medium [RPMI (Gibco, Waltham, MA, USA) plus 1% penicillin-streptomycin (Sigma-Aldrich, St Louis, MO, USA)] referred to as the unstimulated control or with LSDV Neethling strain, BEFV virus or with BHK-21 cells at a 1:10 dilution in medium. Blood was seeded in 96-well plates in triplicate with a total volume of 200 µL/well. Blood was incubated for 24 h in a humidified, 5% CO 2 incubator at 37 • C and brefeldin A was added during the last 5 h of incubation. Red blood cells were removed by lysis using BD Pharm Lyse™ lysing solution (BD Biosciences, Franklin Lakes, NJ, USA), cells were fixed with 4% paraformaldehyde, perforated (0.05% Saponin) and then stained with anti-CD4-FITC (1:50 dilution; BioRad, Hercules, CA, USA); anti-CD8-PE (BioRad, 1:20 dilution) and Mouse anti-Bovine Interferon Gamma:Alexa Fluor ® 647 (1:100 dilution; BioRad, Hercules, CA, USA). Samples were assayed on a FC 500 Beckman Coulter flow cytometer and data analysed using Kaluza version 2.1 (Beckman Coulter, Brea, CA, USA). Values significantly higher than unstimulated control (p ≤ 0.05) were considered positive. The 21 dpc analyses data only included anti-CD8 antibodies and IFNγ due to a shortage of anti-CD4 antibody.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). A parametric t-test or non-parametric Mann-Whitney U test were used for the comparison between two different vaccine groups. Analysis for the unpaired multiple comparison between different time points within a single vaccine group or different vaccine groups at a single time point was performed using Welch one-way analysis of variance (ANOVA) with Dunnett's T3 multiple comparison test or Kruskal-Wallis test with Dunn's multiple comparison test. The paired multiple comparison between different time points within a single vaccine group was performed using repeated measures ANOVA test with Tukey's multiple comparison test. p-values less than 0.05 were considered statistically significant.

Ethics
Authorization to grow LSDV in eggs was granted by the University of Cape Town Animal Ethics committee, (018/012). Ethics approval to test the candidate vaccines in rabbits was granted from the University of Cape Town (AEC 018_039), Stellenbosch University (UCT-DOUG-2019) and the South African Department of Agriculture, Forestry and Fisheries (DAFF), ref: 12/11/1/7/1. Ethics approval for the cattle experiment to be performed at the ARC institute-Onderstepoort Veterinary Research (Transboundary Animal Diseases) was granted by the South African Department of Agriculture, Land Reform and Rural Development (DALRRD), study number: TADP-S-20/02, [DALRRD Ref no: 12/11/1/1].

Construction and Confirmation of Candidate Vaccines against LSDV and BEFV
Two variants of the Neethling vaccine strain of LSDV, nLSDV∆SOD-UCT and nLSDVSODis-UCT [29], were used as parent viruses in the construction of candidate dual vaccines against LSD and BEF. Figure 2 shows, diagrammatically, the design of the LSDV recombinants, whereby the foreign gene cassette was inserted between LSDV open reading frames (ORFs) 49 and 50, which are highly conserved and transcriptionally convergent [41]. The two recombinants, LSDV(∆SOD)BEFV-Ga and LSDV(SODis)BEFV-Ga, were made to express the BEFV G protein gene derived from an Australian BEFV sequence (Ga) together with the red fluorescent protein, mCherry, as a marker. LSDV(∆SOD)BEFV-Gb and LSDV(SODis)BEFV-Gb expressed a BEFV G protein gene derived from a consensus South African sequence (Gb) as well as the green fluorescent protein (eGFP) as a marker. together with the South African Gb protein and eGFP. All G protein genes were expressed from the vaccinia virus mH5 promoter (PmH5) and the M gene was expressed from a modified fowlpoxvirus promoter (PmFPV). The six recombinants were confirmed to be correct by PCR ( Figure 3) and Sanger sequencing of the gene cassette inserted between LSDV ORFs 49 and 50. BEFV gene expression was verified by immunofluorescence ( Figure 4).
Ga, were made to express the BEFV G protein gene derived from an Australian BEFV sequence (Ga) together with the red fluorescent protein, mCherry, as a marker. LSDV(∆SOD)BEFV-Gb and LSDV(SODis)BEFV-Gb expressed a BEFV G protein gene derived from a consensus South African sequence (Gb) as well as the green fluorescent protein (eGFP) as a marker. LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M expressed the matrix (M) protein together with the South African Gb protein and eGFP. All G protein genes were expressed from the vaccinia virus mH5 promoter (PmH5) and the M gene was expressed from a modified fowlpoxvirus promoter (PmFPV). The six recombinants were confirmed to be correct by PCR ( Figure 3) and Sanger sequencing of the gene cassette inserted between LSDV ORFs 49 and 50. BEFV gene expression was verified by immunofluorescence ( Figure 4).

Figure 2.
Diagrammatical representation of six dual LSDV-BEFV candidate vaccines constructed. The foreign gene cassettes were inserted into either nLSDV∆SOD-UCT or nLSDVSODis-UCT, between LSDV ORFs 49 and 50. A BEFV G protein sequence was derived from an Australian isolate (Ga) or a South African consensus sequence (Gb). M = Matrix protein gene; mCherry and eGFP encode red and green fluorescent marker proteins, respectively. The positions of primer binding sites (for = forward and rev = reverse) are shown, as well as the PCR product sizes amplified from these primers. Diagrammatical representation of six dual LSDV-BEFV candidate vaccines constructed. The foreign gene cassettes were inserted into either nLSDV∆SOD-UCT or nLSDVSODis-UCT, between LSDV ORFs 49 and 50. A BEFV G protein sequence was derived from an Australian isolate (Ga) or a South African consensus sequence (Gb). M = Matrix protein gene; mCherry and eGFP encode red and green fluorescent marker proteins, respectively. The positions of primer binding sites (for = forward and rev = reverse) are shown, as well as the PCR product sizes amplified from these primers.

Neutralizing Antibody Responses Elicited by LSDV-BEFV Vaccine Candidates in a Rabbit Model
The candidate vaccines were initially tested in a small animal model (rabbit) to determine which BEFV immunogens and LSDV vaccine backbones to take forward into cattle, a permissive host for LSDV.

Comparison of Different LSDV Backbones with the Same BEFV (Gb-M) Gene Inserts
A second experiment was performed in rabbits, following the same procedure, to compare the two different LSDV vector backbones with the BEFV Gb and M gene inserts ( Figure 6). All rabbits, inoculated with either LSDV(∆SOD)BEFV-Gb-M or LSDV(SODis) BEFV-Gb-M, elicited positive neutralizing responses to both LSDV and BEFV, but no difference could be observed between the two groups (p = 0.1825 for the BEFV neutralization titres and p = 0.318 for the LSDV neutralization titres; Mann-Whitney test). Because rabbits are non-permissive to LSDV growth, it was hypothesized that a stronger response would be elicited in a bovine host, and a difference in the host response to the LSDV vector backbone may be observed, due to the presence or absence of the SOD gene homologue.

Testing of LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M in Cattle
Two groups (n = 10) of cattle were vaccinated twice, four weeks apart, with homologous LSDV(∆SOD)BEFV-Gb-M or LSDV(SODis)BEFV-Gb-M vaccines and monitored daily for clinical signs of disease. Neither of the groups of cattle showed clinical signs of LSD, other than a raised temperature on day 1 post vaccination, which returned to normal the next day. There was no rise in temperature following the boost vaccination, given 32 days post initial vaccination. During the course of the experiment, two animals (ID 109 and 128), in the LSDV(∆SOD)BEFV-Gb-M group, died prior to the boost vaccination and another four (ID 120, 121, 104 and 116), two from each group, died prior to the challenge at day 201. All deaths were unrelated to the vaccinations. Three animals died as a result of acute, haemorrhagic fibrinonecrotic pneumonia, probably caused by environmental stress. These were animals 128 (died on day 19 post vaccination), 104 (died on day 59 post vaccination) and 121 (died on day 117 post vaccination). Two animals succumbed to complications as a result of bloat. These were animals 109 (died on day 35 post vaccination) and 116 (died on day 200 post vaccination). Both animals had recurrent bloat and had been treated on several occasions before they died.  The two parent LSDV vaccines (nLSDV∆SOD-UCT and nLSDVSODis-UCT) were used as negative controls. Anti-B-Phemeral rabbit serum was used as the primary antibody for all samples (1:1000 dilution). (a) detection of BEFV Ga expression (green) using donkey antirabbit Alexa488 secondary antibody (1:500); (b) detection of BEFV Gb (red) and BEFV-Gb-M (red) using anti-rabbit CY3 secondary antibody (1:500). Nucleic acid was stained with Hoechst (blue).

Neutralizing Antibody Responses Elicited by LSDV-BEFV Vaccine Candidates in a Rabbit Model
The candidate vaccines were initially tested in a small animal model (rabbit) to determine which BEFV immunogens and LSDV vaccine backbones to take forward into cattle, a permissive host for LSDV.
(a) (b) (c) Figure 5. Comparison of dual vaccines expressing different BEFV gene inserts in a rabbit model. Rabbits were divided into groups of 5, and each animal was inoculated intramuscularly with 10 6 ffu of the respective vaccines LSDV(∆SOD)BEFV-Ga, LSDV(∆SOD)BEFV-Gb and LSDV(∆SOD)BEFV-Gb-M. nLSDV∆SOD-UCT was given to a group of 4 animals as a negative control for BEFV. Rabbits were given three doses of homologous vaccine at 28-day intervals and neutralization was tested on serum taken 14 days post final inoculation. Neutralization titres are expressed as the reciprocal of the dilution required to neutralize virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. (a) graph showing individual animal responses to both BEFV (blue) and LSDV (orange). BEFV (blue) and LSDV (orange) responses to the dual vaccines, expressing different BEFV inserts, were compared in (b,c) respectively. Statistical analysis was conducted using the Kruskal-Wallis test with Dunn's multiple comparison test. Horizontal lines indicate median values. * p < 0.05, ** p < 0.01.  LSDV(SODis)BEFV-Gb-M, elicited positive neutralizing responses to both LSDV and BEFV, but no difference could be observed between the two groups (p = 0.1825 for the BEFV neutralization titres and p = 0.318 for the LSDV neutralization titres; Mann-Whitney test). Because rabbits are non-permissive to LSDV growth, it was hypothesized that a stronger response would be elicited in a bovine host, and a difference in the host response to the LSDV vector backbone may be observed, due to the presence or absence of the SOD gene homologue. Figure 6. Comparison of two different LSDV vector backbones with the same BEFV gene inserts. Rabbits (n = 5 per group) were given three inoculations, 28 days apart, of either LSDV(∆SOD)BEFV-Gb-M or LSDV(SODis)BEFV-Gb-M, intramuscularly, at a dose of 10 6 ffu per rabbit. Blood was taken 14 days after the final inoculation and serum was tested for neutralization of BEFV and LSDV. Neutralization titres are expressed as the reciprocal of the dilution required to neutralize virus in 50% or more of wells of cells infected with BEFV (blue) or LSDV (orange), respectively.

Testing of LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M in Cattle
Two groups (n = 10) of cattle were vaccinated twice, four weeks apart, with homologous LSDV(∆SOD)BEFV-Gb-M or LSDV(SODis)BEFV-Gb-M vaccines and monitored daily for clinical signs of disease. Neither of the groups of cattle showed clinical signs of LSD, other than a raised temperature on day 1 post vaccination, which returned to normal the next day. There was no rise in temperature following the boost vaccination, given 32 days post initial vaccination. During the course of the experiment, two animals (ID 109 and 128), in the LSDV(∆SOD)BEFV-Gb-M group, died prior to the boost vaccination and another four (ID 120, 121, 104 and 116), two from each group, died prior to the challenge at day 201. All deaths were unrelated to the vaccinations. Three animals died as a result of acute, haemorrhagic fibrinonecrotic pneumonia, probably caused by environmental stress. These were animals 128 (died on day 19 post vaccination), 104 (died on day 59 post vaccination) and 121 (died on day 117 post vaccination). Two animals succumbed to complications as a result of bloat. These were animals 109 (died on day 35 post vaccination) and 116 (died on day 200 post vaccination). Both animals had recurrent bloat and had been treated on several occasions before they died. The increases in neutralization responses, against both BEFV and LSDV, were significantly higher for both groups of animals after the boost vaccination ( Figure 7a,b and Figure 8a,b, respectively). The differences between these responses in the two groups of animals were not statistically significant (Figure 7c,d). All vaccinated animals retained BEFV neutralization antibodies up until 169 dpb (Figure 7a,b) with no significant difference between the two groups at 169 dpb ( Figure 7e).

Neutralization Responses to BEFV and LSDV
Although LSDV neutralization titres did not differ significantly between the two groups at 14 and 30 dpb (Figure 8a-d), there was a notable difference at day 201, when the group vaccinated with LSDV(∆SOD)BEFV-Gb-M showed no detectable neutralization response, whereas 7/8 of the animals from the group vaccinated with LSDV(SODis)BEFV-Gb-M had positive LSDV neutralization responses (Figure 8e). Following challenge, all animals mounted rapid and strong LSDV neutralization responses by 14 dpc, which remained at the same level at 28 dpc (Table 2 and Figure 8a,b,f,g). In comparison, the three naïve control animals developed increases in neutralizing antibody titres from 14 to 28 dpc (Table 2 and Figure 8f,g). Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = pre-immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals which developed lesions at sites of inoculation (sizes of lesions in brackets); only 4/20 and 7/20 animals developed neutralizing antibodies against BEFV and LSDV, respectively. Following the boost vaccination, all animals developed neutralizing responses to both BEFV and LSDV. Neutralizing antibodies against BEFV persisted for >6 months post boost in all vaccinated animals. All animals vaccinated with LSDV(∆SOD)BEFV-Gb-M lost detectable LSDV neutralizing antibodies by day 201 (169 dpb), but 7/8 of the animals vaccinated with LSDV(SODis)BEFV-Gb-M retained LSDV neutralizing antibodies until the time of challenge (169 dpb). le 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the tion required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The er the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = preunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals ch developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).    eutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the equired to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The e titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = preation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals veloped lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).  Table 2 shows the neutralizing responses elicited at different time points post vaccination, post booster vaccination and post LSDV challenge. After one inoculation, only 4/20 and 7/20 animals developed neutralizing antibodies against BEFV and LSDV, respectively. Following the boost vaccination, all animals developed neutralizing responses to both BEFV and LSDV. Neutralizing antibodies against BEFV persisted for >6 months post boost in all vaccinated animals. All animals vaccinated with LSDV(∆SOD)BEFV-Gb-M lost detectable LSDV neutralizing antibodies by day 201 (169 dpb), but 7/8 of the animals vaccinated with LSDV(SODis)BEFV-Gb-M retained LSDV neutralizing antibodies until the time of challenge (169 dpb). able 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the ilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The igher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = premmunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals hich developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).  Vaccines 2021, 9, x Table 2 shows the neutralizing responses elicited at differ vaccination, post booster vaccination and post LSDV challenge. A only 4/20 and 7/20 animals developed neutralizing antibodies aga respectively. Following the boost vaccination, all animals de responses to both BEFV and LSDV. Neutralizing antibodies against months post boost in all vaccinated animals. All anima LSDV(∆SOD)BEFV-Gb-M lost detectable LSDV neutralizing antibo dpb), but 7/8 of the animals vaccinated with LSDV(SODis)BEFVneutralizing antibodies until the time of challenge (169 dpb). Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as t dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; aster which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated t   Table 2 shows the neutralizing responses elicited at vaccination, post booster vaccination and post LSDV challe only 4/20 and 7/20 animals developed neutralizing antibodie respectively. Following the boost vaccination, all anima responses to both BEFV and LSDV. Neutralizing antibodies ag months post boost in all vaccinated animals. All LSDV(∆SOD)BEFV-Gb-M lost detectable LSDV neutralizing dpb), but 7/8 of the animals vaccinated with LSDV(SODis)B neutralizing antibodies until the time of challenge (169 dpb). Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are express dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regard immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unre     Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are e dilution required to neutralize the virus in 50% or more of wells of cells infected with BE higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post cha which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died    eutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the equired to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The e titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = preation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals veloped lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).   Table 2 shows the neutralizing responses elicited at different time points post vaccination, post booster vaccination and post LSDV challenge. After one inoculation, only 4/20 and 7/20 animals developed neutralizing antibodies against BEFV and LSDV, respectively. Following the boost vaccination, all animals developed neutralizing responses to both BEFV and LSDV. Neutralizing antibodies against BEFV persisted for >6 months post boost in all vaccinated animals. All animals vaccinated with LSDV(∆SOD)BEFV-Gb-M lost detectable LSDV neutralizing antibodies by day 201 (169 dpb), but 7/8 of the animals vaccinated with LSDV(SODis)BEFV-Gb-M retained LSDV neutralizing antibodies until the time of challenge (169 dpb). able 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the ilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The igher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = premmunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals hich developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).   Table 2 shows the neutralizing responses elicited at differ vaccination, post booster vaccination and post LSDV challenge. A only 4/20 and 7/20 animals developed neutralizing antibodies aga respectively. Following the boost vaccination, all animals de responses to both BEFV and LSDV. Neutralizing antibodies against months post boost in all vaccinated animals. All anima LSDV(∆SOD)BEFV-Gb-M lost detectable LSDV neutralizing antibo dpb), but 7/8 of the animals vaccinated with LSDV(SODis)BEFVneutralizing antibodies until the time of challenge (169 dpb). Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as t dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; aster which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated t   Table 2 shows the neutralizing responses elicited at vaccination, post booster vaccination and post LSDV challe only 4/20 and 7/20 animals developed neutralizing antibodie respectively. Following the boost vaccination, all anima responses to both BEFV and LSDV. Neutralizing antibodies ag months post boost in all vaccinated animals. All LSDV(∆SOD)BEFV-Gb-M lost detectable LSDV neutralizing dpb), but 7/8 of the animals vaccinated with LSDV(SODis)B neutralizing antibodies until the time of challenge (169 dpb). Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are express dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regard immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unre     Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are e dilution required to neutralize the virus in 50% or more of wells of cells infected with BE higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post cha which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died     sponses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the alize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The er the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = pres post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).    zation responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The he darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = prev = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).            Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. T higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = p immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show anim which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the recip dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respe higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pr immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks sh which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccin    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as t dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; aster which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated t    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are express dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regard immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unre    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are e dilution required to neutralize the virus in 50% or more of wells of cells infected with BE higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post cha which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died       sponses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the alize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The er the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = pres post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).   zation responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The he darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = prev = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).           Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = preimmunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. T higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = p immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show anim which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the recip dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respe higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pr immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks sh which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccin  Table 2 shows the neutralizing responses elicited at differ vaccination, post booster vaccination and post LSDV challenge. A only 4/20 and 7/20 animals developed neutralizing antibodies aga respectively. Following the boost vaccination, all animals de responses to both BEFV and LSDV. Neutralizing antibodies against months post boost in all vaccinated animals. All anima LSDV(∆SOD)BEFV-Gb-M lost detectable LSDV neutralizing antibo dpb), but 7/8 of the animals vaccinated with LSDV(SODis)BEFVneutralizing antibodies until the time of challenge (169 dpb). Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as t dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; aster which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated t    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are express dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regard immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unre    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are e dilution required to neutralize the virus in 50% or more of wells of cells infected with BE higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post cha which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died   eutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the equired to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The e titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = preation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals veloped lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination). Titre  0  14  28  46  62  201  229  0  14  28  46  62  201 215 229 pre-im 14 dpv 28 dpv 14 dpb 30 dpb 169 dpb 197 dpb pre-im 14 dpv 28 dpv 14 dpb 30 dpb 0 dpc 14 dpc 28 dpc      Titre  ay  0  14  28  46  62  201  229  0  14  28  46  62  201 215 229 pre-im 14 dpv 28 dpv 14 dpb 30 dpb 169 dpb 197 dpb pre-im 14 dpv 28 dpv 14 dpb 30 dpb 0 dpc 14 dpc 28 dpc    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as t dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; aster which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated t  Table 2 Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are express dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regard immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unre  Table 2 Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are e dilution required to neutralize the virus in 50% or more of wells of cells infected with BE higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post cha which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died   eutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as the reciprocal of the equired to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV, respectively. The e titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as -ve. pre-im = preation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; asterisks show animals veloped lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated to vaccination).    Vaccines 2021, 9, x Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are expressed as t dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or LSDV higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regarded as immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge; aster which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unrelated t    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are express dilution required to neutralize the virus in 50% or more of wells of cells infected with BEFV or higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were regard immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post challenge which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died (unre    Table 2. Neutralization responses of cattle to BEFV (blue) and LSDV (orange). Titres are e dilution required to neutralize the virus in 50% or more of wells of cells infected with BE higher the titre the darker the shade. Titres < 1/5 for BEFV and <1/4 for LSDV were immunisation, dpv = days post vaccination, dpb = days post boost, dpc = days post cha which developed lesions at sites of inoculation (sizes of lesions in brackets); ☨ animal died  14 and 30 dpb, were tested for BEFV binding antibodies, using pre-immune sera as a negative control (OD 450 < 0.2) and a kit positive control (OD 450 > 0.3). The ELISA plates were coated with a BEFV G protein monomer of Asian origin, which included the conserved linear epitope 1. All animals produced binding antibodies at 14 and 30 dpb (Figure 9a,b), with no significant differences between the groups vaccinated with LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M.

Protection of Vaccinated Cattle from LSDV Challenge
The cattle vaccinated with LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M were challenged with a dose of 10 7 TCID 50 virulent LSDV at day 201 (almost 6 months post boost). A control group of three naïve animals was also challenged in the same way. All control animals developed localized swelling at the site of injection at 4 dpc and a raised temperature for over a week post infection. Figure 10a shows the control animals at 14 dpc and Figure 10b shows their rectal temperatures taken over a period of 6 weeks (two weeks prior to challenge and 4 weeks post challenge). In addition, the control group of animals became sensitive to touch from day 4 post infection and this lasted for approximately 10 days. Both groups of vaccinated animals developed a fever one day post challenge (data not shown), but temperatures subsided after one day. No other signs of illness were presented, showing that the vaccinated animals were protected against LSD.
The increases in neutralization responses, against both BEFV and LSDV, were significantly higher for both groups of animals after the boost vaccination (Figures 7a,b  and 8a,b, respectively). The differences between these responses in the two groups of animals were not statistically significant (Figure 7c,d). All vaccinated animals retained BEFV neutralization antibodies up until 169 dpb (Figure 7a,b) with no significant difference between the two groups at 169 dpb (Figure 7e). Although LSDV neutralization titres did not differ significantly between the two groups at 14 and 30 dpb (Figure 8a-d), there was a notable difference at day 201, when the group vaccinated with LSDV(∆SOD)BEFV-Gb-M showed no detectable neutralization response, whereas 7/8 of the animals from the group vaccinated with LSDV(SODis)BEFV-Gb-M had positive LSDV neutralization responses (Figure 8e). Following challenge, all animals mounted rapid and strong LSDV neutralization responses by 14 dpc, which remained at the same level at 28 dpc (Table 2 and Figure 8a,b,f,g). In comparison, the three

T Cell Responses to BEFV and LSDV Post LSDV Challenge
Cattle challenged with virulent LSDV were tested for CD4 + and CD8 + T cell responses prior to challenge (0 dpc) and, at weekly intervals, post LSDV challenge (7, 14 and 21 dpc). Whole blood was stimulated with either LSDV or BEFV. naïve control animals developed increases in neutralizing antibody titres from 14 to 28 dpc (Table 2 and Figure 8f,g).

Binding Antibody Responses to BEFV
Sera, from time points 14 and 30 dpb, were tested for BEFV binding antibodies, using pre-immune sera as a negative control (OD450 < 0.2) and a kit positive control (OD450 > 0.3). The ELISA plates were coated with a BEFV G protein monomer of Asian origin, which included the conserved linear epitope 1. All animals produced binding antibodies at 14 and 30 dpb (Figure 9a,b), with no significant differences between the groups vaccinated with LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M. Horizontal lines indicate median values. * p < 0.05, ** p < 0.01, *** p < 0.001.

Protection of Vaccinated Cattle from LSDV Challenge
The cattle vaccinated with LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M were challenged with a dose of 10 7 TCID50 virulent LSDV at day 201 (almost 6 months post boost). A control group of three naïve animals was also challenged in the same way. All control animals developed localized swelling at the site of injection at 4 dpc and a raised temperature for over a week post infection. Figure 10a shows the control animals at 14 dpc and Figure 10b shows their rectal temperatures taken over a period of 6 weeks (two weeks prior to challenge and 4 weeks post challenge). In addition, the control group of animals became sensitive to touch from day 4 post infection and this lasted for approximately 10 days. Both groups of vaccinated animals developed a fever one day post challenge (data not shown), but temperatures subsided after one day. No other signs of illness were presented, showing that the vaccinated animals were protected against LSD.

T Cell Responses to BEFV and LSDV Post LSDV Challenge
Cattle challenged with virulent LSDV were tested for CD4 + and CD8 + T cell responses prior to challenge (0 dpc) and, at weekly intervals, post LSDV challenge (7, 14 and 21 dpc). Whole blood was stimulated with either LSDV or BEFV.
T Cell Responses to BEFV Post Challenge Vaccinated cattle challenged with virulent LSDV did not develop CD4 + T cell responses to BEFV (data not shown). However, a significant CD8 + T cell response, as compared to control stimulants, was detected at 7 dpc in 5/8 animals and in two animals at 21 dpc (animal ID 105 and 122) tested in the group vaccinated with LSDV(SODis)BEFV-

Discussion
Despite the availability of vaccines against BEF and LSD, these two diseases remain a threat to the cattle industry. Several different platforms have been used to make BEFV vaccines [10], with inactivated and live attenuated vaccines being the most widely used. Inactivated BEFV vaccines require multiple doses and, even then, are not fully protective [42,43]. A combination of live attenuated followed by killed vaccine [44] is used in Japan [11], the vaccines being based on local strains of BEFV. The live attenuated B-Phemeral, derived from an African BEFV isolate, is used in South Africa in a two-dose regimen [12].

Discussion
Despite the availability of vaccines against BEF and LSD, these two diseases remain a threat to the cattle industry. Several different platforms have been used to make BEFV vaccines [10], with inactivated and live attenuated vaccines being the most widely used. Inactivated BEFV vaccines require multiple doses and, even then, are not fully protective [42,43]. A combination of live attenuated followed by killed vaccine [44] is used in Japan [11], the vaccines being based on local strains of BEFV. The live attenuated B-Phemeral, derived from an African BEFV isolate, is used in South Africa in a two-dose regimen [12].
The G protein is recognized as being antigenic [11,[13][14][15][16] and has been used as a protein vaccine [13]. Secreted forms of the BEFV G protein, lacking the transmembrane portion of the protein, are being investigated as subunit vaccines [45]. Virus vectors, such as rabies [46], Newcastle Disease virus [40], vaccinia virus [14] and LSDV [47], which express the BEFV G protein, continue to be explored as platforms for BEFV vaccine development. Proof of concept that a recombinant poxvirus could be used to protect cattle is given by Hertig et al., (1996) who demonstrated that the vaccinia virus expressing BEFV G protein could protect cattle from BEFV challenge [14]. Since then, LSDV has been explored as a vector for novel BEFV vaccines [47,48]. All previous research on poxvirus recombinants expressing BEFV proteins is based on the Australian BEFV G protein [14,47,48]. The LSDV-BEFV recombinants were made many years ago, using the LSDV Neethling vaccine as a for boosting responses to the transgene product [52]. However, multiple immunizations with LSDV did not result in prevention of infection with the boosting virus. The challenge experiment would indicate that neutralizing responses played a role in controlling infection together with T cell responses. This is an indication that LSDV could be used to deliver multivalent vaccines, or alternatively, different vaccines at different times without the immunity to LSDV preventing infection with the recombinant virus.
The recombinant vaccines described in this paper are potential candidates for dual vaccines against LSD and BEF. The two recombinants LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M warrant further testing in the field as they were both safe at the dosage tested, which was 10 times higher than the standard LSDV vaccine dose. The cattle used in this experiment were Friesian dairy cattle, which were expected to be the most sensitive cattle to LSDV vaccines. No evidence of Neethling associated disease was observed. However, the vaccines need to be tested in different species of cattle to further determine dosage, immunogenicity and safety. We hypothesize that the recombinants expressing the BEFV Ga protein would be more suited to use in Asia and Australia; and those vaccines expressing BEFV Gb would be more suitable for use in Africa.

Conclusions
Six candidate vaccines have been developed to simultaneously immunize cattle against BEF and LSD. In a rabbit model, all vaccines elicited neutralizing responses to the live attenuated B-Phemeral vaccine strain of BEFV, including the vaccine candidates expressing the Australian BEFV Ga gene. With Africa in mind, greater focus was on those vaccines based on the consensus African sequence of BEFV (Gb) as vaccines expressing the Gb protein are likely to be more protective in the African setting. It could not conclusively be shown that the BEFV M gene improved immunogenicity, but the highest neutralization titres were produced by rabbits vaccinated with LSDV expressing both Gb and M protein genes. The two candidate vaccines, LSDV(∆SOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M, elicited neutralizing responses to both BEFV and LSDV in cattle. In addition, they conferred protection against virulent LSDV challenge, inducing rapid, strong neutralizing responses post challenge as well as CD4 + and CD8 + T cell responses.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.