Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses

Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs.

and tongue lesions, cough, diarrhea, nasal and ocular discharge, and depression are typical clinical PPR disease signs. The PPR disease etiological agent is Peste des Petits Ruminants Virus (PPRV), a single-stranded negative sense enveloped RNA virus belonging to Paramixoviridae family, Morbillivirus genus (1) whose genome contains six genes coding for eight proteins. Among these, Hemagglutinin (H) is a structural glycoprotein with hemagglutinin and neuraminidase activities, involved in host cell targeting and virus attachment. H glycoprotein is an immunodominant antigen which, alone, can stimulate a protective immune-response when delivered by several viral vectors, mainly based on adenovirus (2)(3)(4) and poxvirus (5,6). These antigen immune-properties would allow the generation of a Differentiating Infected from Vaccinated Animals (DIVA) vaccine. Since PPRV H glycoprotein is the only PPRV antigen expressed by the viral vector, the use of an ELISA against a different antigen, such as PPRV nucleo-capsid protein (N), would allow to distinguish naturally infected animals from vaccinated animals. Viral vectors are not only simply delivery systems but they can also work as adjuvants, unspecificaly stimulating the immune system and therefore increasing the specific active/ protective immunity. Different classes of viruses have been tested as viral vectors and each presents particular advantages and disadvantages, depending on their biological characteristics and on the host, who needs to be protected toward a specific disease. Hence, it is arduous to predict which viral vector could be the best. A specific viral-vector should be able to confer selective immunization only against a specific pathogen and not toward others. Consequently, it would be of great interest to explore new vector vaccines based on different viruses. Bovine herpesvirus 4 (BoHV-4) is a dsDNA genome virus belonging to Herpesviridae family, Gammaherpesvirus sub-family and Rhadinovirus genus. BoHV-4 natural host is cattle, whereas its best experimental host is the rabbit. However, BoHV-4 has been isolated from domestic and non-domestic bovine species such as African buffalo (Syncerus caffer) (7), American bison (Bison bison), or zebus (Bos indicus) and small ruminants such as sheep and goats (8). Some feline isolates from lions (9) and cats (10) were also reported. Moreover, BoHV-4 isolates were also obtained from the kidney of an apparently healthy monkey (Aotus trivirgatus) (11). BoHV-4 can replicate in vitro in primary cultures and cell lines from a variety of animal species (12)(13)(14)(15)(16)(17)(18), whereas in vivo, it can experimentally infect mice (16,19,20), rats (21), rabbits (15), sheep (13), swine (22), and goats (18). Moreover, ex vivo non-human primate tissue explants infections have also been observed (paper in preparation). Another BoHV-4 important feature, which makes it an attractive gene delivery vector, is that in contrast to other gamma herpesviruses, BoHV-4 is not oncogenic and its infection is not directly linked to a specific pathology. Since BoHV-4-based vector has been successfully employed to immunize mice (16,19,20), sheep (13), and goats (18), in the present work, an exploratory immunization study for PPRV in mice, before applying BoHV-4-based vector in sheep and goats, was performed. A recombinant BoHV-4 expressing the PPRV Hemagglutinin gene (Nigeria 75/1 strain) was generated. BoHV-4-A-PPRV-H-ΔTK immunized mice developed both PPRV neutralizing antibodies and PPRV specific T-cell responses. These data indicate that this BoHV-4-based vector could be an effective PPR vaccine candidate for small ruminants that could distinguish between infected and vaccinated animals.

constructs generation
Synthetic PPRV-H ORF was first amplified from pGEM-T Easy-PPRV-H template by PCR using NheI-PPRV-H sense (5′-ccccgctagcccaccatgtccgcacaaagggaaagg-3′) and Phos-PPRV-H antisense (5′-agactggattacatgttacctc-3′) pair of primers in order to insert NheI restriction site at 5′ terminus and a phosphate group at 3′ terminus. The PPRV-H amplicon generated was then cloned into NheI/SalI blunt cut pIgK-E2BVDV3-gD106 intermediate shuttle vector (Clontech) to generate pIgK-PPRV-H-gD106. The gD106 tagged fragment was excised from the intermediate plasmid cutting with NheI and BamHI blunt restriction enzymes to be subsequently cloned inside the pINT2-EGFP final shuttle vector cut with NheI and SmaI restriction enzymes in order to generate pINT2-PPRV-H-gD106.

Bac recombineering and selection
Recombineering was performed as previously described (26) with some modifications. For heat-inducible homolog recombination in SW102 Escherichia coli (E. coli), containing the BAC-BoHV-4-A-TK-KanaGalK-TK genome targeted into the TK locus with KanaGalK selector cassette, the PvuI linearized pTK-CMV-PPRV-H-TK expression cassette was used. 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 (https://redrecombineering. ncifcrf.gov/).

southern Blotting
To further confirm our results, a Southern Blotting with a probe spanning H 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 (14). 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).
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 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-cm 2 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).

Viruses and Viral replication
BoHV-4-A-PPRV-H-ΔTK 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 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 previously described (27). Virus pellets were then resuspended in cold EMEM without FBS. TCID50 were determined on BEK cells by limiting dilution.

Flow cytometry intracellular cytokine staining assays
Splenocytes from inoculated mice were prepared as previously described (24). For responses to PPRV, splenocytes were cultured overnight with BEI-inactivated PPRV (Nig'75) (28). To assess responses to PPRV-H murine T cell epitopes H5 (H(551-559) YFYPVRLNF) and H9 (H(427-441) ITSVFGPLIPHLSGM) (29), splenocytes were expanded in vitro for 1 week with 10 µg/ ml peptide before measuring IFN-γ responses. For intracellular IFN-γ measurements, cells were cultured at 10 6 cells per well in the presence of different stimuli (peptide or PPRV) overnight before the addition of 10 µg/ml brefeldin-A (Sigma) for the last 5 h of incubation. Phorbol myristyl acetate (20 ng/ml) and ionomycin (1 µg/ml) (both from sigma) stimulation was used as positive control for IFN-γ production. Vehicle (DMSO)stimulated (no peptide) or irrelevant peptides (gp33-41 peptide (KAVYNFATC) from lymphocytic choriomeningitis virus) were used as negative control. No differences in background IFN-γ production was detected between these negative control groups. Following stimulation, cells were stained with anti-mouse CD4-FITC and anti-mouse CD8-PerCP antibodies (BDpharmingen). Cells were fixed and permeabilized in PBS containing 4% paraformaldehyde and 0.1% saponin (wt/vol). Cells were then stained with anti-mouse IFN-γ-PE (BD pharmingen) and acquired using a FACSCalibur flow cytometer (Becton Dickinson). Gating strategy is described in Ref. (29). Gating for positive IFN-γ positive events was set using isotype and fluorescence minus one channel controls. Data were analyzed with FlowJo software (TreeStar Inc.).

Flow cytometry cytotoxicity assays
Splenocytes from BoHV-4-A-PPRV-H-ΔTK immunized mice were expanded with H5 peptide for 1 week in vitro. These stimulated splenocytes were used as effector cells. RMA/s target cells were labeled with PKH67 green fluorescent linker as described in Ref. (30) and pulsed with relevant peptide. Vehicle-pulsed (no peptide) RMA/s cells were used as negative control. Effector cells and target cells were incubated for 4 hours at 37°C in 96 U-bottom well plates. Cells were then transferred to FACS tubes, dead cells labeled with propidium iodide (PI) (2 µg/ml), and samples immediately analyzed by flow cytometry. Target cells were gated on bright FL1+ cells. Positive maximum cell death controls (target cells in PBS + 0.2% saponin) and spontaneous cell death controls were used in all experiments. The percentage of specific target cell lysis was calculated following the formula: % specific lysis = 100 × (% PI+ target -% spontaneous death)/(% maximum death − % spontaneous death).

PPrV neutralization assays
Serum samples were inactivated for 30 min at 56°C and tested for the presence of neutralizing antibodies as previously described (31). Briefly, Nigeria 75/1 PPRV stock was incubated with serial dilutions of inactivated sheep serum for 1 hour at RT in triplicate. VDS cells at a concentration of 1.5 × 10 5 cells/ml were added to each well and incubated for 7 days, fixed with 2% formaldehyde and cells visualized by crystal violet staining. Wells without virus served as controls. The plates were monitored for PPRV CPE for 7 days. The VNT titer was defined as the highest dilution of serum that inhibited 50% of the CPE. Sera with VNT titers of 1:10 were considered negative.

statistical analysis
Power analysis (32) was used to determine treatment group size to assess T cell responses and PPRV seroneutralization. Statistical analysis was performed using Prism 5.0 software (Graphpad Software Inc., USA). Mann-Whitney test was used to compare IFN-γ production in CD4+ and CD8+ T cells. Levels of significance were *p < 0.05, **p < 0.01, and ***p < 0.001.

BohV-4-a-PPrV-h-ΔTK immunization induces a specific neutralizing antibody response against PPrV-h
To determine the presence of neutralizing antibodies, sera from vaccinated mice obtained at 28 days post first immunization   Table 1).
No neutralization activity was detected in pre-immune sera or sera from mice injected with either PBS or BoHV-4-A. These results show that in vivo inoculation of recombinant BoHV-4-A expressing the PPRV-H protein is able to induce the production of PPRV neutralizing antibodies, suggesting that this approach has the potential to confer protective immunity to BoHV-4-A-PPRV-H-ΔTK vaccinated animals.

DiscUssiOn
In developing countries, most of the population is engaged in small-scale farming, 80% of these households keep livestock mostly constituted by small ruminants, primarily sheep and goats. Their productivity is constrained by multiple factors, including infectious diseases where PPR represents one of the most important ones. Vaccination can reduce animal mortality, increase milk and meat production, and positively impact on household revenues. As a result, vaccination also contributes to poverty alleviation by increasing household benefits and freeing income for food, healthcare, or child education. Therefore, new effective vaccines that target diseases that hamper farming in developing countries will have great social and economic benefit (33).
For PPRV eradication campaign, a DIVA vaccine would be of great value to facilitate PPRV sero-surveillance programs and speed up strategies for disease control and eradication (34). The most important drawback when a classical live attenuated vaccine is used is the inability to distinguish the immune response stimulated by vaccination from the one induced by a natural infection. A DIVA vaccine would therefore be a smart solution that combines vaccination with sero-surveillance. DIVA vaccine can be applied not only with gene-deleted marker vaccines (35) but also with sub-unit vaccines (36), heterologous vaccines (37), and recombinant vector-based vaccines. With regard to the last case and as an alternative viral vector, a BoHV-4-based vector platform was employed in the present work to deliver and express PPRV-H gene in transduced cells of immunocompetent mice as surrogate animal model. Although no murine model for PPRV induced disease exists, they represent an invaluable model to initially test the immunity induced by new prototype vaccines. The direct use of large animals could represent a major waste of resources, in terms of maintenance and biosafety containment structures, especially in the event of experiment failure. Data provided by immunized mice not only can be obtained quickly and cheaply but also they could represent a predictive and orientative tool of the vaccine immunogenicity in the natural host, e.g., goats and sheep in the specific case of PPRV (4,25). PPRV-H protein possesses both hemagglutinin and neuraminidase activities and has a hydrophobic domain at the N-terminus (amino acid position [35][36][37][38], which remains within the mature protein acting as a signal peptide that anchors the protein into the membrane (38). The presence of N-terminal 34 amino acids located inside the membrane characterizes PPRV-H as a type II glycoprotein (38). Since PPRV-H protein has been shown to be a good candidate antigen (3,4), a recombinant BoHV-4 delivering an optimized PPRV-H expression cassette was constructed in order to test the immunogenicity of this BoHV-4-based vector and exploit it as a DIVA vaccine platform for PPR vaccination. BoHV-4 has no clear direct disease association; however, its pathogenic potential cannot be absolutely excluded. This is an important consideration since it is to be used as a gene delivery vector. In fact, BoHV-4 has been often associated with postpartum metritis in cattle along with specific endometotropic (39,40). The secretion of prostaglandin E2 (PGE2) and then stimulation of viral replication by PGE2, TNF-α, and lipopolysaccharide (LPS) were suggested as a pathogenic model for BoHV-4 and bacterial co-infection in endometritic cows (41)(42)(43). Therefore, a putative non-pathogenic biotype of BoHV-4 (BoHV-4-A) isolated from the milk cell fraction of a healthy cow whose genome was cloned as a bacterial artificial chromosome (pBAC-BoHV-4-A) (14) was employed. Importantly, BoHV-4-A-based vector behaves like a replicating incompetent viral vector in both wild-type and immunocompromised mice, showing complete absence of pathogenicity (16,17,19,27,44,45). PPRV-H ORF was customized under the transcriptional control of the CMV promoter and integrated into BoHV-4-A genome TK locus. The derived replicationdeficient recombinant vector could transduce mammalian cells and expressed PPRV-H protein. This construct could therefore potentially elicit immunity to the transgene. Genetic stability of viral vectors remains a very important issue, since recombinant viral vectors constitute "genetically modified organisms" (GMO). In our case, the BoHV-4-A-PPRV-H-ΔTK construct was stable through several passages. Relevant planning will however be needed before this recombinant vector can legally be licensed for employment in the field.
Protective natural immunity to morbilliviruses requires both humoral and cellular components of the adaptive immune system. Humoral immunity can protect against the prototype morbillivirus measles virus re-infection, whereas cellular immunity controls virus clearance and dissemination (46,47). In the present work, mice immunized with BoHV-4-A-PPRV-H-ΔTK produced CD4+ and CD8+ T cell responses against PPRV-H epitopes and promoted CTL responses against PPRV. This recombinant vector vaccine can therefore potentially stimulate the T cell immunity essential for virus clearance. It will be interesting in future work to determine whether similarly to recombinant adenovirus vaccines (29), BoHV-4-A-PPRV-H-ΔTK immunization can trigger memory T cell responses in PPRV natural hosts.
However, the most striking results were related to the production of virus neutralizing antibodies (VNAs) against PPRV. It was previously shown that a neutralization titer higher than 10 correlates with a long-lasting humoral response and could be considered as a successful vaccination and protection indicator in the field (48,49). In this pilot study, the lowest VNA titer obtained for all vaccinated mice was never below 120. It could thus be speculated that vaccinated BoHV-4-A-PPRV-H-ΔTK animals could be protected from virulent PPRV challenge when this protocol will be/is applied in the natural host. This is further supported by the fact that BoHV-4 has been successfully used in sheep and goats (13,18). BoHV-4-based vector delivering H alone also induced neutralization titers higher than those obtained with other viral vectors delivering both H and F antigens, which is in line with the concept that H glycoprotein of Paramyxovirus is a stronger inducer of VNA than the F glycoprotein (50,51). Despite the notion that antibody immune response against PPRV is the main factor for an efficient protection, cellular immune response can be also important for virus clearance. In some cases, protection has been obtained even with undetectable level of VNA titers (52,53). The high VNA titer levels and the induction of cellular immunity after BoHV-4-A-PPRV-H-ΔTK immunization indicate that this recombinant vector vaccine has the potential to protect from virulent viral challenge. The induction of humoral and cellular immunity after BoHV-4-A-PPRV-H-ΔTK inoculation indicates that this vaccine can trigger PPRV immunity in the natural host both in an experimental setting and in the field.
In conclusion, in the present paper, it was demonstrated that BoHV-4-A-PPRV-H-ΔTK is able to induce a strong specific immune response against PPRV. These findings are paving the way for BoHV-4-A-PPRV-H-ΔTK use as a safe, large, potent, non-integrative, replicating competent viral vector for PPR vaccination and eradication.

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