Background
Ebola virus (EBOV) is a pathogen responsible of outbreaks of human hemorrhagic fever in African countries, including the last epidemic, which ended with more than 11,300 deaths in Guinea, Liberia and Sierra Leone (updated since September 20th;
http://apps.who.int/ebola/ebola-situation-reports). EBOV infection is characterized by systemic viral replication, host immunity hyper responsiveness along with a cytokine storm and disseminated intravascular coagulation similar to septic shock [
1]. EBOV belongs to the
Filoviridae family which includes two genera, Ebolavirus and Marburgvirus. The genus
Ebolavirus includes three species pathogenic in humans,
Zaire ebolavirus [case fatality report (CFR) 70–90%];
Sudan ebolavirus (CFR ~50%) and
Bundibugyo ebolavirus (CFR ~25%) [
2].
Vaccine production and availability is widely dependent on commercial factors. Indeed, it is not a mere coincidence if vaccines dedicated to important diseases of undeveloped countries are less prevalent on the market than those for diseases of developed countries. An important exception could be represented by EBOV vaccines. Although the disease has been known by the scientific community since 1976, an effective, commercially available vaccine is still lacking. The recent EBOLA outbreak, which began in December 2013, affected both people in isolated rural areas and in large cities. The outbreak reached global dimensions and EBOV-infected patients have been hospitalized not only in Africa but also in USA and Europe. This phenomenon captured the attention of the global scientific community. However, research activity in this field is hampered by the need of costly facilities which is the most important issue in dealing with infectious pathogens for which there are few available vaccines and no effective treatment.
So far a dozen vaccines proved effective protection in non-human primates from lethal EBOV infection and several ones are in advanced trial phases. Most of these vaccine approaches are viral vector-based, where the immune-dominant full length membrane glycoprotein (GP) open reading frame is delivered by a recombinant viral vector. Platforms based on recombinant adenovirus serotype 5 (rAd5) vectors [
3], combined DNA/rAd5 vectors [
4], combined rAd serotype 26 and 35 vectors, recombinant chimpanzee adenovirus serotype 3 (rChAd3) vectors [
3], alphavirus replicons based on recombinant human parainfluenza virus 3 (rHPIV3) [
5], rabies virus [
6], and recombinant vesicular stomatitis virus (sVSV) [
7], have been exploited with successful results [
8]. Vectorialized viruses are not only mere delivery systems but also a sort of adjuvant which strongly induce an active immunity. There are several types of viral vectors, derived from different classes of viruses and each of them possess particular characteristics. It is therefore difficult to predict which virus will best achieve the vaccine-vector goal. It must be kept in mind that a specific viral-vector could be suitable for the immunization toward a specific pathogen, but not toward others. Consequently, it would be of great interest to explore new vaccine-vector agents based on different viruses. Bovine herpesvirus 4 (BoHV-4)-is a relatively new viral vector derived from bovine
gammaherpesvirus. Recombinant BoHV-4s cloned as bacterial artificial chromosome (BAC), delivering ORFs coding for immune-dominant antigens from different pathogens, were shown to successfully elicit a functional immune-response in mice [
9,
10], rats [
11], rabbits [
12], sheep [
13], swine [
14] and goats [
15]. In the present paper, a BoHV-4-based vector platform was generated exploiting a synthetic gene approach; a recombinant BoHV-4 delivering EBOV GP ORF expression cassette was constructed and goats were successfully immunized.
Methods
Cell lines
Bovine embryo kidney [(BEK) were from Dr. M. Ferrari, Istituto Zooprofilattico Sperimentale, Brescia, Italy; (BS CL-94)], BEK expressing cre recombinase (BEK cre) [
16] and human embryo kidney 293T [(HEK 293T) ATCC: CRL-11268] cell lines were cultured in complete growth medium Eagle’s minimal essential medium (EMEM, LONZA) containing 10% fetal bovine serum (FBS), 2 mM of
l-glutamine (SIGMA), 100 IU/mL of penicillin (SIGMA), 100 μg/mL of streptomycin (SIGMA) and 2.5 μg/mL of Amphotericin B (SIGMA) and incubated at 37 °C, 5% CO
2.
Constructs
Synthetic Zaire Ebola virus Mayinga glycoprotein (GP) ORF, tagged at the carboxy-terminal with gD106 peptide (syEBOVgD106) was excised from pUC57sy EBOVgD106 (EUROFINS, GENOMICS) with
NheI and
SmaI enzymes and the 2153 bp fragment was inserted inside
NheI/
SmaI cut pINT2EGFPTK shuttle vector [
17] to generate pINT2CMVsyEBOVgD106. EBOV secreted fragment (EBOVsec), without the trans-membrane domain, was obtained by amplification from pINT2CMVsyEBOVgD106 with
NheI EBOGP sense (5′- ggggctagcccaccatgggcgtg-3′) and
SalI EBOGP antisense (5′-ggggtcgacctggcgccagccggtccaccagtt 3′) primers. The generated 1967 bp
NheI-EBOsec-
SalI was inserted inside
NheI/
SalI digested pIgkE2gD106 to obtain the pCMVEBOsecgD106 construct.
Transient transfection
Confluent HEK 293T cells were seeded into six well plates (3 × 105 cells/well) and incubated at 37 °C with 5% CO2. When the cells were sub-confluent, the culture medium was removed and the cells were transfected with pINT2CMVsyEBOVgD106 using polyethylenimine (Pei) transfection reagent (POLYSCIENCES, INC.). Briefly, 3 μg of DNA were mixed with 7.5 μg PEI (1 mg/mL) (ratio 1:2.5 DNA-Pei) in 200 μL of Dulbecco’s modified essential medium (DMEM) high glucose (EUROCLONE) without serum. After 15 min at RT, 800 μL of medium without serum were added and the transfection solution was transferred to the cells and left for 6 h at 37 °C with 5% CO2, in a humidified incubator. The transfection mixture was then replaced with fresh medium (EMEM, with 10% FBS, 50 IU/mL of penicillin, 50 μg/mL of streptomycin and 2.5 μg/mL of Amphotericin B) and incubated for 24 h at 37 °C with 5% CO2.
Viruses and viral replication
BoHV-4-syEBOVgD106ΔTK and BoHV-4-A were propagated by infecting confluent monolayers of BEK cells at a multiplicity of infection (MOI) of 0.5 tissue culture infectious doses 50 (TCID50) per cell and maintained in medium with only 2% FBS for 2 h. The medium was then removed and replaced with fresh EMEM containing 10% FBS. When the cytopathic effect (CPE) affected the majority of the cell monolayer (~72 h post infection), the virus was prepared by freezing and thawing cells three times and pelleting the virions through a 30% sucrose cushion, as described previously [
18]. Virus pellets were then resuspended in cold EMEM without FBS. TCID
50 were determined with BEK cells by limiting dilution.
Semi-reducing western immunoblotting
Protein cell extracts were obtained from a six-well confluent plate of HEK 293T transfected with pINT2CMVsyEBOVgD106 and from 25-cm2 confluent flasks of BEK infected with BoHV-4- syEBOVgD106ΔTK by adding 100 μL of cell extraction buffer (50 mM Tris–HCl, 150 mM NaCl, and 1% NP-40; pH 8). A 10% SDS-PAGE gel electrophoresis was used to analyze cell extracts containing 50 μg of total protein, after protein transfer in nylon membranes by electroblotting, the membranes were incubated with primary bovine anti BoHV-1 glycoprotein D monoclonal antibody (clone 1B8-F11; VRMD, Inc., Pullman, WA, USA), diluted 1:15.000, and then with a secondary antibody probed with horseradish peroxidase-labelled anti-mouse immunoglobulin (SIGMA), diluted 1:10.000, to be visualized by enhanced chemiluminescence (ECL KIT; PIERCE). Cell supernatant obtained from HEK 293T transfected with pCMVEBOsecgD106 was collected at different time points (16, 24, 40, 48, 50, 60 and 70 h after transfection) and analyzed as above.
BAC recombineering and selection
Recombineering was performed as previously described [
19] with some modifications. After recombineering, only those colonies that were kanamycin negative and chloramphenicol positive were kept and grown overnight in 5 mL of LB containing 12.5 mg/mL of chloramphenicol. BAC DNA was purified and analyzed through
HindIII restriction enzyme digestion. DNA was separated by electrophoresis in a 1% agarose gel, stained with ethidium bromide, and visualized through UV light. Original detailed protocols for recombineering can also be found at the recombineering website (
http://recombineering.ncifcrf.gov).
Southern blotting
DNA from 1% agarose gel was capillary transferred to a positively charged nylon membrane (ROCHE), and cross-linked by UV irradiation by standard procedures [
16].
The membrane was pre-hybridized in 50 mL of hybridization solution (7% SDS, 0.5 M phosphate, pH 7.2) for 1 h at 65 °C in a rotating hybridization oven (TECHNA INSTRUMENTS). The 1967 bp amplicon for EBO digoxigenin-labeled probe was generated by PCR with
NheI EBOGP sense (5′-ggggctagcccaccatgggcgtg-3′) and
SalI-EBOGP antisense (5′-ggggtcgacctggcgccagccggtccaccagtt 3′) primers, as previously described [
12].
Cell culture electroporation and recombinant virus reconstitution
BEK or BEK cre cells were maintained as a monolayer with complete DMEM growth medium with 10% FBS, 2 mM l-glutamine, 100 IU/mL penicillin and 10 mg/mL streptomycin. When cells were sub-confluent (70–90%) they were split to a fresh culture flask (i.e., every 3–5 days) and were incubated at 37 °C in a humidified atmosphere of 95% air–5% CO2. BAC DNA (5 μg) was electroporated in 600 μL DMEM without serum (EQUIBIO APPARATUS, 270 V, 960 mF, 4-mm gap cuvettes) into BEK and BEK cre cells from a confluent 25-cm2 flask. Electroporated cells were returned to the flask, after 24 h the medium was replaced with fresh medium, and cells were split 1:2 when they reached confluence at 2 days post-electroporation. Cells were left to grow until the appearance of CPE. Recombinant viruses were propagated by infecting confluent monolayers of BEK cells at a M.O.I. of 0.5 TCID50/cell and maintaining them in EMEM with 10% FBS for 2 h.
Viral growth curves
BEK cells were infected with BoHV-4-A and BoHV-4syEBOVgD106ΔTK at a M.O.I. of 0.1 TCID50/cell and incubated at 37 °C for 4 h. Infected cells were washed with serum-free EMEM and then overlaid with EMEM containing 10% FBS, 2 mM l-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin and 2.5-mg/mL Amphotericin B. The supernatants of infected cultures were harvested after 24, 48, 72 and 96 h, and the amount of infectious virus was determined by limiting dilution on BEK cells.
Samples collection and ELISA procedure
Blood samples were processed for ELISA test. Briefly, microplates (MICROLON HIGH BINDING) were coated overnight at 4 °C with 50 ng/well EBOsecgD106 protein supernatant obtained from 175-cm
2 sub-confluent HEK 293T cells, transfected with pCMVEBOsecgD106 (Additional file
1: Figure S1) and diluted in 0.1 M carbonate/bicarbonate buffer pH 9.6. After blocking with 1% bovine serum albumin (BSA), serum samples at different dilutions (1/10, 1/100, 1/1000 and 1/10,000) were incubated for 1 h at room temperature. After three washing steps, 50 μL of donkey anti-goat IgG-HRP (SANTA CRUZ BIOTECNOLOGY, Germany) diluted 1:1.000 was added to each well and the plate was incubated as above. Following the final washing step, the reaction was developed with 3,3′,5,5′-tetramethylbenzidine (TMB), stopped with 0.2 M H
2SO
4 and read at 450 nm.
Discussion
In this work, the potential utility of BoHV-4 as a safe, potent, large-capacity gene delivery vector for EBOV antigen was shown. A workflow strategy to construct a BoHV-4-based vector was generated and it was able to deliver an immune-dominant antigen derived from a BSL4 pathogen, thus avoiding all the economical and safety requirements necessary for the manipulation of this kind of biological agent. Furthermore, it was able to elicit a strong humoral immune-response. The results were obtained through a synthetic gene approach, currently based on solid-phase DNA synthesis, which allows the complete synthesis of a double-stranded DNA molecule with no apparent limits in nucleotide sequence or size.
As a model pathogen, EBOV was the primary choice. Firstly because a severe Ebola outbreak was taking place in African countries at the beginning of this project and at that time no approved prophylactic or therapeutic protocols were available. Secondly, EBOV it is classified as a Category A priority pathogen by NIAID and a Category A agent of bioterrorism by the CDC.
The EBOV genome is a single negative-strand RNA molecule encoding seven structural proteins, among which EBOV GP is a type I transmembrane glycoprotein of 676 amino acids in length and its transcript is made by an unusual transcriptional editing [
23]. Full length GP has been shown to be responsible for eliciting a protective humoral immune response in infected individuals [
8]. The cleavage of surface GP, by cellular metalloprotease, tumor necrosis factor α-converting enzyme (TACE), generates shed GP [
21], which contributes to the host protective immune response [
22]. Firstly, in silico customized full length GP ORF was successfully expressed in eukaryotic cells with a suitable expression vector and then integrated in BAC BoHV-4 genome through homologous recombination. BoHV-4-syEBOVgD106ΔTK replicated in cell culture at the same extent as the parental virus, thus no detrimental effect induced by the topological location of the foreign DNA in the BoHV-4 genome was observed. BoHV-4 is a
Gammaherpesvirus belonging to the
Rhadinovirus genera. Although BoHV-4 natural host is cattle, the virus has been isolated from other ruminants, including zebu (
Bos indicus), American bison (
Bison bison), African buffalo (
Syncerus caffer), and sheep. Like other
Herpesviruses, BoHV-4 is able to establish persistent infection in cells of the monocyte/macrophage lineage [
24,
25] and in a bovine macrophage cell line (BoMAC) [
26]. Furthermore, BoHV-4 experimental inoculation in rabbit demonstrates how spleen, as well as macrophages, are the main site of viral persistence [
27]. Due to the lack of a direct correlation between BoHV-4 infection and specific lesions or pathology, BoHV-4 is not considered a primary pathogen and its genome was cloned as bacterial artificial chromosome (BAC), in order to be exploited as a gene delivery vector for immunization purposes and oncolysis. BoHV-4-based vector delivering antigens have been employed to immunize mice [
9], rats [
11], rabbits [
12], sheep [
13], swine [
14], cows (paper in preparation) and goats [
15] without any associated clinical signs or pathology.
Although goats are not susceptible to EBOV infection, BoHV-4-syEBOVgD106ΔTK immunization was performed in adults goats as they have previously been shown to be an appropriate large animal model for BoHV-4-based vector immunization [
15]. Further, goats could be exploited as a source of antibodies production for antibody-based therapeutic in post-exposure treatment of EBOV disease. A pair of newly published studies [
28,
29], demonstrated the efficacy of an ovine polyclonal antibody therapy against EBOV disease when tested in the stringent guinea pig model of EBOV disease.
The sub-cutaneous route of BoHV-4-based vector administration was shown to facilitate antigen production and vector replication takes place only at the site of inoculation [
15], without spreading to the rest of the animal body. No BoHV-4-syEBOVgD106ΔTK viremia was detected in inoculated animals, although all BoHV-4-syEBOVgD106ΔTK inoculated animals were successfully immunized and high titers of EBOV GP antibodies were detected. The ability of BoHV-4-syEBOVgD106ΔTK to efficiently transduce goat skin cells, which consecutively expressed large amounts of GP, explains the consistent titer of antibodies produced and detected by the ELISA assay.
Whether the antibodies induced by BoHV-4-syEBOVgD106ΔTK in goats serum, correlates with a potential protection following a passive transfer in a suitable stringent model of EBOV disease remains unknown. Based on previous studies, which demonstrated the ability of BoHV-4 based vectors to efficiently protect against the Category A agent Monkeypox virus [
10], it can be assumed that the use of BoHV-4 vector based platform represents an effective tool to test unknown antigens and vectors for class A pathogens.
Authors’ contributions
DG conceived the experiments. AR, SJ, GT, ST, VF, FM and DG performed the experiments. SC and GK contributed with reagents. GD and DPK analysed the data. GD wrote the paper. All authors read and approved the final manuscript.