Bovine embryo kidney [(BS CL-94) BEK, from M. Ferrari, Istituto Zooprofilattico Sperimentale, Brescia, Italy], Dubai Camel cells (ATCC^® CRL-2276™, Dubca), BEK-expressing cre recombinase (BEK cre) (3), human embryo kidney 293T [(HEK293T) ATCC: CRL-11268], and alpaca skin stromal cells (23) were cultured in Eagle’s minimal essential medium (EMEM, Lonza) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine (SIGMA), 100 IU/mL penicillin (SIGMA), and 100 µg/mL streptomycin (SIGMA) and 2.5 µg/mL of Amphotericin B (SIGMA) and incubated at 37 C, 5% CO₂. R5-SupT1-transducted cell line are CCR5-expressing cell lines referred to as SupT1-R5 clone M10 (medium expression of CCR5). The clone was obtained by engineered SupT1 cells and kindly provided by H. Garg (24). The cells (5 × 10⁵) were propagated in complete medium and supplemented with 3 µg/mL of Blasticidin (Calbiochem, Germany).

The hCCR5 ORF, was excised from (the commercial plasmid) pcDNA3-hCCR5 (kindly provided by DR. Toon Laeremans, University of Bruxells) cutting with MluI, blunt ended and SmaI restriction enzymes. The ~2,900 bp hCCR5 fragment was then cloned into SmaI cut pINT2 shuttle vector (2) to generate pTK-CMV-hCCR5-TK.

HEK293T cells were seeded into six well plates (3 × 10⁵ cells/well) and incubated at 37°C with 5% CO₂. When cells were sub-confluent, the culture medium was removed, and the cells were transfected with pTK-CMV-hCCR5-TK using polyethylenimine (PEI) transfection reagent (Polysciences, Inc.). Briefly, 3 µg of DNA was 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 room temperature, 800 µL of medium without serum was added, and the transfection solution was transferred to the cells (monolayer) 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, 100 IU/mL of penicillin, 100 µg/mL of streptomycin, and 2.5 µg/mL of Amphotericin B (SIGMA) and incubated for 24 h at 37°C with 5% CO₂.

The PvuI linearized pTK-CMV-hCCR5-TK was used for heat-inducible homologous recombination in SW102 E. coli containing the BAC-BoHV-4-A-TK-KanaGalK-TK genome targeted to the TK locus with KanaGalK selector cassette. Recombineering was performed as previously described (25) 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. Selected SW102 E. coli clones carrying pBAC-BoHV-4 recombinants were analyzed by HindIII restriction enzyme digestion. Original detailed protocols for recombineering can also be found at the recombineering website (https://redrecombineering.ncifcrf.gov/background-info/what-is-recombineering.html).

To further confirm our results, a Southern blotting with a probe spanning human CMV promoter sequence, was performed. DNA from 1% agarose gel was capillary transferred to a positively charged nylon membrane (ROCHE), and cross-linked by UV irradiation by standard procedures (3). The membrane was pre-hybridized in 50 mL of hybridization solution (7% SDS, 500 mM sodium phosphate, pH 7.2) for 1 h at 65°C in a rotating hybridization oven (TECHNA INSTRUMENTS).

CMV probe labeled with digoxigenin was generated by PCR with CVM-KpnI-sense (5′-caggggtacctagttattaatagtaatcaat-3′) and CVM-KpnI-antisense (5′-cgcgggtaccgctagcggatctgacggttca 3′) primers, as previously described (4). The PCR amplification reaction was carried out in a final volume of 50 µL, containing 10 mM Tris–hydrochloride pH 8.3, 5% dimethyl sulfoxide, 0.2 mM deoxynucleotide triphosphates, 2.5 mM MgSO₄, 50 mM KCl, and 0.25 µM of each primer. One hundred nanograms of DNA were amplified over 35 cycles, each cycle consisting of 1 min of denaturation at 94°C, 1 min of primer annealing at 55°C, and 1 min of chain elongation with 1 U of Pfu DNA polymerase (Fermentas) in addition to 1 µL of digoxigenin-11-dUTP, alkali-labile (Roche Life Science) at 72°C.

Bovine embryo kidney 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 100 µg/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 cytopathic effect (CPE).

Bovine herpesvirus 4 (BoHV-4)-CMV-hCCR5ΔTK and BoHV-4-A were propagated by infecting confluent monolayers of BEK cells at a multiplicity of infection 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 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 (26). Virus pellets were then resuspended in cold EMEM without FBS. TCID50 were determined with BEK cells by limiting dilution.

Bovine embryo kidney cells were infected with BoHV-4-A and BoHV-4-CMV-hCCR5ΔTK at a MOI 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, and 72 h, and the amount of infectious virus was determined by limiting dilution on BEK cells. Viral titer differences between each time point are the averages of triplicate measurements ± SEs of the means (P > 0.05 for all time points as measured by Student’s t-test).

To evaluate the presence of hCCR5 protein on the cell surface, a flow cytometry assay was performed on pTK-CMV-hCCR5-TK-transfected HEK293T cells. Cells expressing the protein of interest and mock-transfected cells, which served as negative control, were both assayed with a FITC mouse-antihuman CD195 monoclonal antibody capable of binding to CCR5 Membrane Protein (Clone 2D7/CCR5, Catalog No. 555992, BD Pharmingen™).

The cells plated in a T75 cm² were transfected with 22.5 µg of pTK-CMV-hCCR5-TK DNA, 67.5 µg of PEI (ratio 1:3 DNA-PEI) in 1.5 mL of DMEM high glucose (Euroclone) without serum. After 15 min at room temperature, 6 mL of medium without serum were added, and the transfection solution was transferred to the cells (monolayer) and left for 6 h at 37°C with 5% CO₂, in a humidified incubator. After 6 h, the transfection mixture was then replaced with fresh medium EMEM, with 10% FBS.

The following day, the transfected cells were firstly briefly washed with sterile phosphate-buffered saline (PBS) to remove any traces of serum and subsequently detached with a PBS–ethylenedinitrilotetraacetic (EDTA) solution (50 µL of EDTA acid 500 mM in a final volume of 50 mL). 2 × 10⁵ resuspended cells, for every sample to test, were centrifuged at 1,200 rpm for 4 min at R.T.

The pelleted cells were incubated at R.T. for 20 min with FITC mouse-antihuman CD195 monoclonal antibody, 1:40 diluted in a final volume of 200 µL of PBS–FBS 2% as suggested by BD Pharmingen. Moreover, 1 MOI of BoHV-4-CMV-hCCR5ΔTK was used to infect BEK, DUBCA, and alpaca cells checking the in vitro protein expression on the surface of infected cells by flow cytometry as described before.

Cells surface binding was also performed on Sup-T1-CCR5-M10 cells as previously described (27). Briefly, cells were incubated with 1:10 diluted serum in PBS with 3% FBS from pre-immunized and post-immunized rabbits for 1 h at 4°C, then the cells were washed with PBS and incubated with PE-conjugated goat anti-rabbit antiserum (Sigma-Aldrich) for 30 min at 4°C. Surface expressed CCR5 molecules were detected with anti-CCR5 monoclonal antibody 2D7-PE as the positive control (PE-CF594, mouse-antihuman CD195 monoclonal antibody Becton Dickinson, CA, USA), which has given a mean fluorescence of 40.03. Negative control of binding was done using 5 × 10⁵ cells incubated with PE-conjugated goat anti-rabbit antiserum (Becton Dickinson) for 30 min at 4°C. Ten thousand gated events were acquired using a FACSCalibur flow cytometer (Becton Dickinson). Live cells initially gated by forward and side scatter were analyzed by mean fluorescence intensity (MFI). MFI of each sample was calculated by subtracting MFI of negative control. The results are given as mean of MFI of two independent experiments.

Rabbits were cared for and used in accordance with Italian laws for animal experimentation. Rabbits were maintained at 24°C with a controlled light cycle (12 h of light, starting at 6:00 a.m.) and with food and water ad libitum. Blood samples were obtained, and viral injections were performed via the auricular vein at scheduled intervals. In agreement with the current legislation on animal experimentation, which suggests to minimize the number of animals employed, two adult rabbits, after collection of the preimmune serum, were inoculated intravenously with 1 mL of 10⁵ TCID50 of BoHV-4-CMV-hCCR5ΔTK. A second inoculation with an identical dose of BoHV-4-CMV-hCCR5ΔTK was done 2 weeks apart from the first inoculation. Blood samples were collected at 2 weeks, just before the second inoculum, and 5 weeks from the first inoculum. The experiments comply with the Principles of Animal Care (NIH Publication no. 85-23. Revised 1985) of the National Institutes of Health and with the current law of the European Union and Italy. The present project was approved by the Ethical Committee of the University of Parma (OPBA: prot. n. 49/13 del 08/07/2013).

To evaluate the presence of anti-CCR5 antibody, an immunofluorimetric assay was performed on pTK-CMV-hCCR5-TK-transfected and mock-transfected HEK293T cells and treated with the serum of BoHV-4-CMV-hCCR5ΔTK-vaccinated rabbits.

The cells plated in a T75 cm² were transfected with 22.5 µg of pTK-CMV-hCCR5-TK DNA, 67.5 µg of PEI (ratio 1:3 DNA-PEI) in 1.5 mL of DMEM high glucose (Euroclone) without serum. After 15 min at room temperature, 6 mL of medium without serum was added, and the transfection solution was transferred to the cells (monolayer) and left for 6 h at 37°C with 5% CO₂, in a humidified incubator. After 6 h, the transfection mixture was then replaced with fresh medium EMEM, with 10% FBS.

24 h after the transfection, the cells were firstly briefly washed with sterile PBS to remove any traces of serum and subsequently detached with sterile PBS. After three PBS washes, the cells were resuspended in PBS plus rabbit serum diluted to 1:20; preimmune and 5 weeks post vaccination serum were tested. After 1 h at R.T. incubation, cells were washed in PBS three times, and the cells were resuspended in PBS plus 1:40 FITC-conjugated goat anti-rabbit IgG (F0382, SIGMA) secondary antibody. After 30 min of incubation in the dark at 4°C, the samples were PBS washed three times, resuspended in PBS and visualized on a plastic chamber slide with the fluorescence microscope Axiovert S100 (Zeiss). Images were achieved using a digital CCD camera, and images were processed using the AxioVision 40-V4.6.3.0 (Carl Zeiss, Imaging Solution) software program.

Peptides were synthesized by the solid-phase Fmoc method (28) using an Applied Biosystems model 433A peptide synthesizer. After peptide assembly, resin-bound peptides were deprotected as previously described (29) and purified to greater than 95% purity by semipreparative reverse-phase high-performance liquid chromatography. To obtain conformationally restricted etherocyclic peptides, an extra cysteine was added at peptides covering ECL1 extracellular regions as previously described (19–21). All extracellular regions of CCR5 have been synthesized as shown in Table 1 and in particular amino-terminal region from aa 14 to 34 as it has been demonstrated to be immunogenic whereas peptide from 1 to 13 did not induce antibodies (20, 21); ECL1 as constrained peptide, as its linear form did not elicit antibodies (19–21); ECL2 and ECL3 regions in their linear sequence.

To quantify the rabbit serum antibodies against CCR5, microwell plates were coated with synthetic peptides covering the extra membrane regions of CCR5 (amino-terminal, ECL1, ECL2, and ECL3 as shown in Table 1), at 0.1 µg/well in 50 mM carbonate buffer, pH 9.5, for 1 h at 37°C. The plates were saturated for 1 h with 1% milk (Sigma-Aldrich, MO, USA) in PBS. Serial dilutions (from 1:20 up to 1:4,860 by threefold dilutions) of pre-immunized and post-immunized specific antisera were added to 1% milk and 0.1% Tween 20 (Sigma-Aldrich) in PBS. Then, peroxidase-conjugated goat anti-rabbit total IgG (Sigma-Aldrich) was added and incubated for 30 min at 37°C. The enzymatic reaction was developed with the TMB Microwell Peroxidase Substrate System (KPL, MD, USA) and read in a plate reader (Biotek, VT, USA) at 492 nm. The reciprocal endpoint titers were determined as the last sample dilution that produced a threefold greater absorbance than the ones of the sample diluent and expressed at serum dilution 1/n. Two independent experiments have been performed, and results are expressed as mean value of titers. To quantify antibodies directed to conformational epitope of ECL1, avidin (Sigma-Aldrich) was coated at 1.5 µg/well in 50 mM carbonate buffer, pH 9.5 and then biotynilated-ECL1 constrained peptide was used. The rest of the assay was similar as for the ELISA performed with the other CCR5 peptides but using 10% bovine serum albumin (Sigma-Aldrich), 30% fetal calf serum from Lonza, Belgium, and 0.1% Tween 20 in PBS as either blocking or diluent buffer (19).