Vaccine candidate based on a baculovirus expressed VP2 provides full protection from epizootic hemorrhagic disease virus serotype 8 in calves

Abstract

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) has recently emerged in Europe, causing widespread outbreaks in cattle and wild ruminant populations. The lack of commercial vaccines at the time of emergence highlighted the urgent need to develop effective vaccines for disease control.

Therefore, we developed and evaluated the safety and efficacy of a novel subunit candidate vaccine against EHDV-8, based on the recombinant VP2 protein (rVP2) from EHDV-8 expressed in a baculovirus system.

Ten Holstein-Friesian calves were randomly assigned to vaccinated (n = 5) or control (n = 5) groups. Animals in the vaccinated group received two doses of 200 μg of rVP2 protein 21 days apart, while control animals were sham-vaccinated. Fourteen days after the booster dose, both groups were challenged with a field strain of EHDV-8. Clinical signs were recorded daily, and blood samples were collected at regular intervals to assess viremia and immune response.

The rVP2 vaccine candidate was well tolerated, with only transient, low-grade fever and mild local reactions. Vaccinated calves developed neutralizing antibodies by day 28 post-vaccination and, upon EHDV-8 challenge on day 35 post-vaccination, showed no clinical signs or viremia. In contrast, all control calves developed viremia, mild clinical signs, and EHDV-specific antibodies after challenge. Notably, vaccinated animals remained negative by VP7 ELISA even post-challenge, indicating that the rVP2-based vaccine afforded protection from viral replication and disease and enabled DIVA (Differentiating Infected from Vaccinated Animals) capability.

In conclusion, these results demonstrate that the rVP2-based subunit vaccine confers complete protection from EHDV-8 under experimental conditions in cattle, indicating the possibility of its use for disease control strategies.

1. Introduction

Epizootic hemorrhagic disease (EHD) is a non-contagious, Culicoides-borne viral disease of domestic and wild ruminants, included in the list of the notifiable diseases of both the World Organization for Animal Health (WOAH) and the European Animal Health Law as per its possible impact on livestock industry.

The causative agent is the epizootic hemorrhagic disease virus (EHDV), a member of the genus Orbivirus, family Sedoreoviridae, officially designated as Orbivirus ruminantium. At least seven serotypes (1, 2 and 4–8) have been identified globally [1]. The EHDV virion has a genome composed of 10 segments of double-stranded RNA (S1–S10), encased in a double-layered capsid composed of structural proteins VP2, VP3, VP5 and VP7 [1,2]. Among these, the VP2 is the outermost capsid protein and, as such, it is the primary site for virus attachment and the major determinant inducing serotype-specific neutralizing antibodies.

Given its immunodominance and DIVA (differentiating infected from vaccinated animals) capability, the VP2 is the first-choice antigen for monovalent or multivalent vaccine formulations, enabling vaccination without compromising disease surveillance.

Historically, EHDV has been circulating endemically in Africa, the Americas, Australia, Asia and the Middle east [3]. Recently, EHD has gained increasing attention due to the emergence and spread of EHDV serotype 8 (EHDV-8) in southern Europe [4].

The first European outbreaks were reported in October 2022 in domestic and wild ruminants in Italy and Spain, likely resulting from wind-borne introduction of infected Culicoides midges from North Africa [5,6]. The virus subsequently spread through the Iberian Peninsula, reaching France in 2023 and continuing its progression across these regions. This incursion into continental Europe raised significant concerns, due to its extensive spread and capacity to potentially cause clinical disease in cattle populations and significant economic loss for the livestock sector [3,7,8].

Despite its possible detrimental effects on the livestock industry, at the time of the first European outbreaks, no commercial vaccines against EHDV-8 were available in the European market, significantly hampering control efforts and contributing to the virus’s extensive spread. This triggered intensive research and development activities aimed at producing safe and effective vaccines. A key early initiative was led by the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM) in Teramo, Italy, which produced and tested the efficacy of an inactivated EHDV-8 vaccine candidate (vEHDV8-IZSAM) [9].

In parallel, pharmaceutical companies began developing vaccines based on different platforms, that later received limited marketing authorizations under different regulatory pathways. The Zendal Group, via its animal health division Vetia, launched Hepizovac, an inactivated adjuvanted vaccine based on EHDV-8 strain SPA 2022/LCV_03 which is the only vaccine approved for marketing throughout the European Union (EU) (marketing authorization No. EU/2/25/341/001–003). Based on an inactivated whole virus, it does not allow DIVA (Differentiating Infected from Vaccinated Animals) capability.

Nearly at the same time, the Laboratorios Syva developed a recombinant VP2 subunit vaccine with DIVA capability which is a key asset for disease surveillance, especially in countries where vaccination of susceptible hosts is implemented. This vaccine has been licensed for the use in cattle and deer in Spain and Belgium, following a national marketing authorization according to article 110 [2] of Regulation (EU) 2019/6.

The availability of safe, effective, DIVA-compatible and widely licensed EHDV-8 vaccines is expected to significantly influence the future epidemiological landscape of EHD, mitigating its impact on European livestock and supporting coordinated control strategies.

In this context, subunit vaccines including VP2 represent a particularly promising strategy, offering strong immunogenicity, DIVA compatibility, and the potential for use in future multivalent vaccine approaches.

In this study, we tested the safety, immunogenicity and efficacy of an adjuvanted EHDV-8 recombinant VP2 protein (EHDV-8 rVP2) through a controlled vaccination/challenge trial in calves.

2. Materials and methods

2.1. Ethical statement

The animal study was approved and overseen by the Animal Care and Welfare Board of the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM), and authorized by the Italian Ministry of Health (authorization no. 65/2024-PR), in accordance with the Italian Legislative Decree of 4 March 2014, No. 26 - Implementation of Directive 2010/63/EU on the protection of animals used for scientific purposes.

Calves were used in the trial following a 5-day acclimatization period.

2.2. Generation of EHDV-8 rVP2 and vaccine formulation

The full length (2965 bp) cDNA VP2-EHDV-8 coding sequence gene (virus isolate EHDV-82022.TE.50459.1.2-GenBank accession number OP897266.1), optimized for protein expression in insect cells and integrated with two tandem Strep tag II sequences at the 3′ end, was cloned into the pCDNA3.1 vector by GeneArt (Thermo Fisher Scientific, Waltham, USA). The cDNA was then directionally subcloned into the pENTR^™/D-TOPO^® vector (Thermo Fisher Scientific, Waltham, USA). The subcloning was assessed by conventional PCR and the amplicon was gel- extracted (Qiagen^® MinElute Gel Extraction Kit). After quantification, the required amount of DNA was Sanger-sequenced to check both insertion and correct orientation (GENEWIZ, South Plainfield, NJ). The pENTR-EHDV-8 VP2/D-TOPO vector was propagated by transforming One Shot^® chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA). Plasmid DNA from selected colonies was purified with QIAprep^® Spin Miniprep kit (Qiagen, Valencia, CA) and transformation was confirmed by conventional PCR using M13 Rev and an EHDV-8 VP2 Fwd primer. The gene of interest was introduced from the plasmid DNA into a BaculoDirect^™ C-Term linearized DNA (containing a C terminal V5– 6× histidine tag) by an LR recombination and following the BaculoDirect^™ expression system (Thermo Fisher Scientific, Waltham, USA). The LR recombination mix was then transfected into adherent Spodoptera frugiperda (Sf9) cells. Recombination was confirmed by conventional PCR using both baculo-DNA polyhedrin Fwd and VP2 Rev or baculo- V5 rev and VP2 Fwd set of primers. Protein expression (expected mass: ≈115 kDa) was confirmed by SDS-PAGE Coomassie and immunoblotting using anti-His and anti-V5 monoclonal antibodies, other than polyclonal anti EHDV-8 and negative bovine sera.

EHDV-8 rVP2 was purified by affinity chromatography using StrepTrap columns (Cytiva, Wilmington, USA), after lysis of cells under native conditions.

Cell pellet was 20:1 resuspended in lysis buffer then 2× freeze-thawed and 6× short (10 s) sonicated. The lysate was centrifuged at 10,000 rpm for 20′ at 4 °C and the supernatant was 0.45 μm filtered and purified through a StrepTrap column driven by AKTA start (GE, Sweden). Purified protein was dialyzed against phosphate-buffered saline.

Purification was confirmed by NuPAGE Coomassie and immunoblotting; protein concentration was quantified with the bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, Waltham, USA) at an absorbance of 562 nm, with a bovine serum albumin (Sigma-Aldrich, Saint Louis, USA) as protein reference standard.

Protein aliquots (≈1 mg/mL) were stored at −80 °C until use. Before administration to animals, two aliquots (corresponding to 2 mg of rVP2) were mixed with 5 mL of Montanide^™ ISA25 (Seppic, France) and 13 mL of sterile PBS and slowly emulsified under magnetic stirrer agitation for 10 min at room temperature.

2.3. Challenge virus

EHDV-8 was isolated from the spleen of a bovine which died during an outbreak in 2022 in Sardinia, Italy (internal ID: EHDV8–2022TE50472). The organ homogenate was passaged once on Kc cells (RRID: CVCL_RW99) followed by two passages on BSR cells (RRID: CVCL_RW96). Following harvest, the TCID₅₀ of the stock virus, as determined by endpoint titration assay, was 5.80 log₁₀ TCID₅₀/mL.

2.4. Animal study

Ten Holstein-Friesian calves, aged 3 to 4 months, were recruited in the study. The animals were housed at the facilities of the IZSAM. Prior to vaccination, all animals were clinically healthy and confirmed negative for EHDV antibodies and RNA.

The calves were randomized into two groups: Group V of 5 vaccinated animals (identified as V1 to V5) and Group C of 5 mock-vaccinated animals (identified as C1 to C5).

Rectal body temperature was measured for all calves from three days before prime and booster vaccination to the day of vaccination (day post vaccination dpv 0 and dpv 21), and the mean temperature during this period was considered as baseline temperature.

On dpv 0 (prime vaccination) and 21 (booster), Group V received 200 μg of EHDV8-rVP2 in 2 mL medium, while Group C received 2 mL of PBS/montanide. The injections were given subcutaneously in the middle of the neck, alternating the side between prime and booster vaccination.

Following each inoculation, the injection site and body temperature were checked daily for 10 days. Temperature increases of ≥1 °C compared to the baseline were considered indicative of fever.

Clotted blood samples were collected from jugular vein on 0, 7, 14, 21, 24, 28 and 31 dpv; whole blood samples were collected on dpv 0 and 21 only.

On dpv 30 (corresponding to day post challenge −5; dpc-5), calves were relocated in temperature-controlled insect-secure isolation unit, with constant access to water and feed. Each group was housed in a separate box. Body temperature was measured rectally from dpc −3 to dpc 0, and the mean temperature was considered baseline.

Two weeks after booster dose (dpv 35/dpc 0), all calves were challenged with 1.5 mL of EHDV-8 inoculum (1 mL subcutaneously and 0.5 mL intradermally; both sides of the neck), approximately equivalent to 6 log₁₀ TCID₅₀.

After challenge, calves were monitored daily by veterinarians, to check for apparent clinical signs consistent with EHDV infection. Rectal temperature was recorded daily for 13 days post-challenge, and an increase of ≥1 °C from dpc 1 onwards compared to the baseline temperature was considered fever. A clinical score was assigned to each animal using pre-defined clinical criteria (Table 1).

Table 1.

Criteria for clinical scoring in cattle following EHDV-8 challenge.

	SCORE
Clinical Sign	0	1	2	3	4
GENERAL APPEARANCE	Normal	Slightly depressed	Weakness. Reacts only if stimulated. Tends to remain isolated	Marked prostration. Poor reactivity to stimuli	Permanent recumbency, no reaction to stimuli
FEEDING	Eats normally	Decreased appetite	Little interest in food	Does not eat	Has not eaten for ≥2 days
NASAL DISCHARGE	Absent	Serous/mild nasal discharge	Mucosal hyperaemia with muco-purulent nasal discharge	Hemorrhages and/or hemorrhagic nasal discharge, crusty lesions of nasal plane	/
UPPER RESPIRATORY TRACT	Normal respiration	Mild, intermittent cough	Frequent but intermittent cough	Continuous cough, even at rest	/
LOWER RESPIRATORY TRACT	Normal respiration	Mild dyspnea	Moderate dyspnea	Severe dyspnea	Severe dyspnea with ‘air hunger’
HEAD/ORAL CAVITY	Normal	Mucosal hyperemia	Pronounced hyperemia and salivation. Erosions	Hyperemia and cyanosis of mucosa, hemorrhages, head edema. Marked salivation	/
OCULAR LESIONS	Absent	Conjunctival hyperemia	Moderate conjunctivitis with ocular discharge	Keratoconjunctivitis, uveitis. Corneal ‘clouding’	/
FOOT LESIONS	Absent	Hyperemia of coronary band and/or interdigital cleft.	Mild or pronounced coronitis and/or mild intermittent lameness	Hemorrhagic lesions. Coronitis with edema, marked lameness. Reluctance to move	Diffuse hemorrhagic lesions, hoof sloughing, severe lameness
TEMPERATURE	≥1 < 1.5 °C above baseline average	≥1.5 to <2.5 °C above baseline average	≥2.5 °C above baseline average	/	/
DEATH					8 POINTS

Whole and clotted blood samples were collected on dpv 35, 38, 42, 45, 49, 52, 57, 64, 71 and 78, corresponding to dpc 0, 3, 7, 10, 14, 17, 22, 29, 36, and 43, and stored at 4 °C until processed (max 3 days).

2.5. Real-time reverse transcription PCR (rRT-PCR) for relative quantification based on Ct values

EHDV-8 RNA was detected according to the methodology described in Portanti et al. (2023) [10] and targeting a 173 bp region of the S2.

Briefly, total RNA was extracted from 200 μL of blood samples using the MagMAXTM CORE Nucleic Acid Purification Kit (Applied Biosystems, St. Austin, TX, USA) on a KingFisherTM Flex Purification System (Thermo Fisher Scientific, MA, USA), following the manufacturer’s instructions.

After denaturation, real-time reverse transcription PCR was performed using TaqMan^™ Fast Virus 1-Step Master Mix (Applied Biosystems) on a QuantStudio 7 Flex real-time PCR system (Applied Biosystems), with the following protocol: 45 °C for 10 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. A sample was considered positive if Ct < 40.

2.6. Serology

All serum samples were tested for serotype-specific neutralizing antibodies using serum neutralization test [11]. After challenge, serum samples were also tested with a commercial VP7 competitive ELISA (ID Screen EHDV competition; Innovative Diagnostics, France).

Neutralizing antibody titres were defined as the reciprocal of the highest dilution of the serum able to neutralize 80–100 % CPE in the wells. The initial dilution of sera was 1:10, and final dilution 1:1280. Negative results were assigned the value “0.”

ELISA results have been reported as competition percentage (S/NC %), using the following formula: (OD of the sample/OD of the negative control) x100. Samples with S/NC% values ≥40 were classified as negative, values >30 and < 40 are considered as doubt, and values ≤30 are classified as positive.

2.7. Statistical analysis

The number of viremic animals, as determined by rRT-PCR, in vaccinated and control groups was compared using a Fisher’s exact test, to assess whether the adjuvanted EHDV8-rVP2 was able to prevent the development of viremia. A p < 0.05 was set as significant level.

3. Results

3.1. Baculovirus-expressed EHDV-8 VP2 protein specifically detected by monoclonal antibodies and bovine sera

Immunoblotting was first performed on cell lysate showing that anti-His and anti-Strep monoclonal antibodies recognized a relevant EHDV-8 rVP2-specific band with a molecular weight (MW) ranging approximately 115 kDa in infected cells (Fig. 1). SDS-PAGE Coomassie staining of the purified EHDV-8 rVP2, tested before administration, revealed a band with the expected MW, as well (Fig. 2a). An additional immunoblotting of the purified protein with anti-His and anti- V5 monoclonal antibodies confirmed the baculovirus-mediated protein expression (Fig. 2b and c).

Fig. 1.

Detection of rVP2 expression in Sf9 cells. Immunoblot analysis of Sf9 cell lysates (P2,P3) showing expression of EHDV-8 rVP2 (expected mass≈ 115 kDa). Membranes were probed with anti-Strep-Tactin antibody conjugated with a fluorescent label (1:1000; IBA, cat. 2–1562–050) and anti-His tag antibody conjugated with HRP (1:2000; Invitrogen, Waltham, USA - product #R931–25). M: Precision Plus Protein^™ Dual Color Standards (Biorad cod. 1,610,374). Ctrl: uninfected cell lysate.

Fig. 2.

Purified EHDV-8 rVP2 (≈115 kDa) analyzed by SDS page, followed by Coomassie staining (2a) and immunoblotting (2b-c). Immunoblots were performed using anti-His tag (product #R931–25) (2b) and anti-V5 tag (product #R961–25) (2c) monoclonal antibodies (1:1000; Invitrogen, Waltham, USA). M: Novex^™ Sharp Pre- Stained Protein Standard (Invitrogen, Waltham, USA).

3.2. Transient fever and localized reactions after vaccination

Following both vaccination and placebo inoculation, all calves developed a fever lasting 3 to 5 days. In addition, a localized reaction at the injection site was observed in all animals, characterized by the formation of nodules with a diameter up to 8 cm, lasting for one week prior to complete resolution. In some animals, the reaction was notably painful upon touch. The overlying skin was intact. Similar adverse effects were observed following booster vaccination.

3.3. Robust EHDV-8 VP2-specific neutralizing antibody response induced in vaccinated calves

All animals were confirmed seronegative before vaccination. Neutralizing antibodies were detected in all vaccinated calves starting from dpv 28, i.e.7 days after booster vaccination, with titres ranging from 20 to 160. At the time of challenge (14 days post-booster vaccination), vaccinated cattle had a neutralizing titre ranging from 40 to 320 (Fig. 3). A VP7 ELISA test performed before prime and booster vaccination revealed negative results (data not shown). Sham-inoculated animals did not develop an immune response following placebo administration.

Fig. 3.

Neutralizing antibody (Nab) response in mock-vaccinated (C) and rVP2-vaccinated (V) calves. Trend of the Nab titre over time. x-axis: day post-vaccination; y-axis: neutralizing antibody titre. NAb titre was defined as the reciprocal of the highest dilution of the serum able to neutralize cytopathic effect in cells. Negative sera were assigned a titre of 0. dpv: day post vaccination. Arrows indicate (from left to right) prime vaccination (dpv 0), booster vaccination (dpv 21) and challenge (dpv 35).

3.4. Sustained neutralizing antibody titres post-challenge without VP7 ELISA positivity in vaccinated calves

After challenge, EHDV antibodies were measured both by competitive VP7-ELISA and serum neutralization. Control cattle seroconverted using the VP7 ELISA on dpc 10 and remained ELISA positive throughout the post-challenge sampling period. Neutralizing antibodies were first detected in three control calves on dpc 10 and in all control calves on dpc 14, with titres ranging from 40 to 1280.

EHDV-8 rVP2-vaccinated animals did not show an anamnestic response following challenge, with neutralizing titres maintaining a steady level throughout sampling period. In both groups, neutralizing titres were detectable until the end of the experiment (dpv 78, Fig. 3). Notably, vaccinated animals never tested positive in VP7 ELISA, not even after challenge (data not shown).

3.5. EHDV-8 rVP2 vaccination prevents viremia and clinical signs in calves

Viral RNA was not detected in the blood of vaccinated animals. In contrast, EHDV-8 RNA-emia was detected in all sham-inoculated calves starting from dpc 3 and throughout the remainder of the sampling period (Group C vs. Group V p = 0.0079), with peak RNA-emia on dpc 7, as measured by Ct value (range 21.3 to 24.3 - Fig. 4). Additionally, animals administered the adjuvanted EHDV-8 rVP2 showed no signs of EHD following challenge. Conversely, control calves exhibited mild clinical signs typical of EHDV infection, including 1-day fever (C1), prescapular lymphadenomegaly (calf C2), oral erosions (Calf C4), depression and dyspnea (calves C1 and C2) (Fig. 5).

Fig. 4.

RNA-emia in mock-vaccinated (C) and rVP2-vaccinated (V) calves over time. x-axis: day post challenge (dpc); y-axis: rRT-PCR Ct values. Ct: cycle threshold. Vaccinated animals remained rRT-PCR-negative throughout the sampling period.

Fig. 5.

Clinical score in mock- and rVP2-vaccinated calves post-challenge. Clinical signs were monitored daily for 610 days post challenge (dpc). Each row represents an individual calf (V1–V5: rVP2-vaccinated; C1–C5: mock-vaccinated). The intensity of the red shading corresponds to the severity of clinical signs, with darker tones indicating higher scores. rVP2-vaccinated animals showed no clinical signs (scores = 0).

4. Discussion and conclusions

The recent emergence of EHDV-8 in southern Europe has posed significant challenges to livestock health and disease management. When EHDV-8 first emerged in Europe, no vaccines were available to combat the disease, and this allowed EHDV-8 to spread rapidly and widely across Spain and southwestern France. Thousands of outbreaks were reported affecting both domestic cattle and wild ruminants [12–14], with important economic consequences whose full extent has yet to be quantified.

While animal movement restrictions and vector control may help mitigate the incidence of outbreaks, vaccination remains a key pillar in disease control, as shown with bluetongue [15–18].

In general, vaccines must be safe and efficacious; in addition, DIVA compatibility would be highly beneficial for disease surveillance programs.

In this context, the development of safe and effective serotype-specific vaccines against EHDV-8 has become a priority.

In this study, we evaluated the safety, immunogenicity and efficacy of a vaccine candidate containing an EHDV-8 recombinant VP2 protein produced in a baculovirus expression system.

Our findings demonstrate that vaccination with adjuvanted EHDV-8 rVP2 fully protects calves from both clinical disease and infection. No RNA-emia was detected in vaccinated animals post-challenge, indicating sterilizing immunity.

The vaccine formulation was well tolerated, with no systemic adverse events observed, suggesting a favourable safety profile. Local reactions at the injection site were minimal, transient and well tolerated by animals. These local reactions are comparable to those reported for commercially available EHDV-8 vaccines and are most likely ascribable to the adjuvant.

Vaccinated animals developed serotype-specific neutralizing antibodies as soon as 7 days following the booster dose, i.e. all animals were antibody-positive at the time of challenge. Following challenge, rVP2-vaccinated calves remained clinically healthy and rRT-PCR-negative, while all unvaccinated control animals developed RNA-emia and all but one exhibited mild clinical signs consistent with EHD. This result underscores the vaccine’s complete efficacy and confirms the protective efficacy of VP2-directed antibodies, in line with previous studies [1,19,20].

A VP7-competitive ELISA conducted post-challenge confirmed the DIVA compatibility of the rVP2 vaccine. In fact, vaccinated animals remained ELISA-negative even after challenge, supporting a robust immune response that not only prevented the disease, but also inhibited efficient viral replication. Accordingly, neutralizing immune response did not show any boost after challenge infection. This result aligns with previous findings in bluetongue studies, where sheep immunized with a VP2-based subunit vaccine did not seroconvert to VP7 after challenge with BTV-4 [21].

The DIVA capability is crucial for disease surveillance, especially in countries where national vaccination campaigns have been implemented.

Compared to the currently marketed vaccines, the EHDV-8 rVP2 vaccine candidate tested here is capable to both prevent viremia and offer DIVA-compatibility under experimental conditions.

As EHD is a vector-borne disease, preventing viremia means stopping onward transmission of the virus.

The EHDV-8 rVP2 subunit vaccine described here represents a promising vaccine candidate. It presents as an ideal vaccine candidate for EHD control, as it protects animals from clinical signs, offers DIVA possibility and, above all, blocks viremia. However, it needs two vaccine shots to induce protective immune responses.

In addition to its efficacy, the rVP2 subunit vaccine offers several practical advantages. The use of a baculovirus expression system allows for scalable production and rapid adaptation, which is crucial for responding to emerging EHDV strains or for developing multivalent vaccines. In this context, EHDV-2- and EHDV-6-specific rVP2-based vaccines have been previously tested for immunogenicity and/or efficacy [19,20].

However, this study has some limitations. First, it was conducted on a limited number of animals under controlled conditions. While the results are nonetheless notable, the robustness of the result would benefit results on a larger cohort or in field situations.

Second, a relatively high antigen dose (200 μg/animal per vaccination) was used in this study compared to other subunit vaccines. Such a dose allowed us to clearly demonstrate that the rVP2 subunit vaccine can fully protect vaccinated animals from viremia following challenge, but at the same time we are aware that this amount of antigen per animal may not be practical for large-scale vaccination campaigns in the field. Future trials with a lower amount of antigen will therefore be carried to optimize the vaccine formulation while ensuring the full protection of cattle from viremia.

Third, the study does not address the duration of protective immunity and the need for annual vaccinations. Understanding the duration of immunity would offer valuable guidance for optimizing vaccination strategies in real-world settings. Also, we did not evaluate the ability of a single-shot vaccination schedule to provide protective immunity. Future studies are planned to assess these critical aspects.

Finally, given the likely role of wild ruminants in the ecology of EHDV [13,14,22,23], evaluating the vaccine’s efficacy in wildlife populations would provide important insights into its potential for broader disease control.

In conclusion, the EHDV-8 rVP2 protein produced and adjuvanted for the use in cattle induced immune responses that resulted in protection from EHD and prevention of viremia in challenged animals. Except for transient hyperthermia, no adverse systemic events were recorded in vaccinated animals. In addition, adjuvanted EHDV-8 rVP2 vaccine showed DIVA compatibility.

The vaccine’s ability to prevent both clinical disease and viremia, combined with its DIVA compatibility, positions it as a highly promising tool for future EHD control and prevention programs.

Data availability

Data will be made available on request.