Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2⁺ mammary cancer

ABSTRACT

The epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of bovine herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes at high levels and elicited significant cellular immune responses in BALB/c mice upon administration via either DNA vaccination or viral infection. In BALB-neuT mice, instead, only the viral construct expressing the membrane-bound chimeric form of Her-2 protein (BoHV-4-RHuT-gD) elicited a humoral immune response that was more intense and earlier-appearing than that induced by DNA vaccination. In keeping with this observation, two administrations of BoHV-4-RHuT-gD effectively protected BALB-neuT mice from tumor formation, with 50% of vaccinated animals tumor-free after 30 weeks from immunization compared to 100% of animals exhibiting at least one palpable tumor in the case of animals vaccinated with the other BoHV-4-HER-2 constructs.

KEYWORDS:

• BoHV-4-based vector
• BALB-neuT mice
• Her-2 oncogene
• HER-2 antibodies
• recombinant BoHV-4s
• vaccination

Abbreviations

ATP	=	adenosine triphosphate
BoHV-4	=	bovine herpesvirus 4
BAC	=	bacteria artificial chromosome
BEK	=	bovine embryo kidney
BSA	=	bovine serum albumin
CFSE	=	carboxyfluorescein-diacetate-succinimidyl ester
CPE	=	cytopathic effect
DMEM	=	Dulbecco's modified eagle medium
ELISPOT	=	enzyme linked immune-spot
EMEM	=	Eagle's minimal essential medium
Fc	=	crystallizable fragment
FBS	=	fetal bovine serum
gD	=	glycoprotein D
HEK	=	human embryo kidney
HER-2	=	epidermal growth factor receptor 2
HSV-1	=	herpes simplex virus 1
IFNγ	=	interferon gamma
IgG	=	immunoglobulin G
LB	=	Luria Bertani's medium
M.O.I.	=	multiplicity of infection
PBS	=	phosphate buffer saline
PEI	=	polyethylenimine
PCR	=	polymerase chain reaction
RPMI	=	Roswell Park Memorial Institute medium
SEM	=	standard error mean
SFU	=	spot forming units
SPC	=	spot-forming cells
TCID50	=	tissue culture infectious dose 50
TK	=	tyrosine kinase.

Introduction

Despite the significant therapeutic improvements achieved in the last decades, breast cancer remains the most important, women-affecting, solid neoplasm worldwide.¹ Over-expression of the HER-2 oncogene, mainly due to gene amplification-based mechanisms^2,3 occurs in ∼15–25% breast cancers, where it has been consistently associated with metastasization propensity, poor prognosis and reduced survival.⁴

Her-2 is a four transmembrane domain tyrosine kinase (TK) receptor. Although it is structurally and functionally well characterized, its specific ligand is still unknown. Her-2 homo/hetero dimerization induces TK domain phosphorylation, thus triggering the activation of multiple signal transduction pathways.^5-7 Among these pathways, those centered on the Ras/Raf mitogen-activated protein kinase and the phosphatidil-inositole-3-kinase are the best characterized. Their deregulated activation is causally involved in cancerous phenotype development and results in altered cellular growth/division, differentiation and adhesion properties.^5-7 For these reasons, Her-2 and its associated pathways are major clinical therapeutic targets. At present, two main classes of molecules are employed in the clinic to target Her-2. The first is represented by humanized monoclonal antibodies (Trastuzumab and Pertuzumab), that by targeting the extracellular portion of the receptor, interfere with Her-2 dimerization thus inducing receptor endocytosis and degradation.^8,9 Importantly, these antibodies can also activate antibody- and complement-mediated cellular cytotoxicity.¹⁰ The second class of therapeutics is composed of synthetic small-molecules that interact with the ATP-binding site of the intracellular TK domain of Her-2 and block receptor phosphorylation/activation, thus preventing downstream signaling events.^1,11 Of note, Trastuzumab and Her-2 TK domain inhibitors have been shown to act synergistically thus paving the way to their combined therapeutic use also in association with traditional endocrine, chemo and radiation therapies.¹

Prompted by the effectiveness of passive immunization relying on anti-Her-2 antibodies, immune-prophylactic, active immunization approaches directed against Her-2 have also been extensively explored in pre-clinical models of mammary cancer.^12,13 However, since Her-2 is a self-tolerated antigen,¹⁴ a major hurdle for these approaches has been the breaking of central and peripheral tolerance.^14-17 Several ways to overcome this problem have been developed,^18-20 including vaccination with hybrid DNA constructs coding for chimeric rat/human Her-2 proteins.^21-23

A very little explored, but potentially promising tolerance breaking/immunization strategy relies on the use of viral-vectored vaccine formulations. In fact, viral vectors can deliver the antigen directly into host cells, thus leading to high-level transgene cellular expression. Key properties of an effective, and potentially translatable, viral vector are safety, the ability to properly present the expressed antigen to the immune system and to remain within host cells long enough to stimulate an effective response. A viral vector apparently meeting these criteria, including ease of manipulation (with the possibility to insert up to 30 kb of foreign DNA) and the ability to confer strong immunogenicity to heterologous antigens, is bovine herpesvirus 4 (BoHV-4). Cattle is the natural host of this virus, but BoHV-4 isolates have been retrieved from other animal species as well. In vitro, BoHV-4 is able to replicate in primary cultures and cell lines from a variety of animal species.^24-30 Experimental infection of many non-natural hosts (mice,²⁸ rats,³¹ rabbits,²⁷ sheep,²⁵ swine³² and goats³⁰) as well as ex vivo tissue explants from non-human primates has been documented (personal communication), suggesting that BoHV-4 is most likely also competent for human cell transduction. In infected mice, BoHV-4 behaves as a replication-incompetent virus³³ that preferentially localizes to cells of the monocyte/macrophage lineage.³⁴ At variance with other gamma-herpesviruses, no evidence for growth-transformation, nor any virus-associated pathology has been reported for BoHV-4 so far. In fact, recombinant BoHV-4s expressing immune-dominant antigens from different pathogens have been successfully employed to immunize genetically modified mice without any detrimental effect, overt clinical sign or pathology correlated to viral vector inoculation.²⁸ Furthermore, a BoHV-4-based vector armed with a Herpes Simplex virus-1 thymidine kinase (HSV-1-TK) gene displayed enhanced oncolytic properties in immune-competent orthotopic syngenic mouse and rat glioma models.²⁹

In view of all these favorable properties, and good potential for clinical translation, we set out to test BoHV-4 as a HER-2 expression carrier and novel immuno-prophylactic agent against Her-2⁺ mammary cancer. Since vaccine delivery and cellular localization of vaccine-encoded antigens are key factors in modulating the induced immune responses, we assembled different recombinant HER-2-BoHV-4 viral vectors and tested their immunogenicity as well as cancer prevention capacity. The recombinant vector expressing the membrane-bound form of a hybrid, rat-human Her-2 antigen was found to be the only one capable of eliciting high anti-Her-2 antibody titers in immune-tolerant, rat HER2 transgenic (BALB-neuT) mice and to afford strong protection against autochthonous Her-2⁺ mammary cancer development in these animals.

Results

Design and expression of different Her-2 chimeric proteins

Before generating BoHV-4-based vectors expressing specific portions of HER-2 oncogene, three optimized ORFs coding for different HER-2 derived chimeric fragments were customized taking into account antigen subcellular localization and recognition by the immune system. RHuT-gD, a cell surface associated form, was assembled by fusing the N-terminal 1–390 aminoacids region of rat HER-2 with 299 amino acids (residues 301–691) derived from the C-terminal region of human HER-2 and gD106, a 33 peptide tag derived from bovine herpesvirus-1 glycoprotein D³⁵ (Fig. S1). RRT-gD, a secreted form lacking the transmembrane domain, was constructed by fusing the N-terminal 1–390 amino acids region of rat HER-2 with the gD106 tag peptide (Fig. S2). An additional secreted form, potentially capable of interacting with Fc receptors and designated RRT-Fc, was generated by substituting the gD106 region of RHuT-gD with a stretch of 240 amino acids derived from the C-terminus of mouse IgG Fc (Fig. S3). RHuT-gD, RRT-gD and RRT-Fc were all placed under the transcriptional control of the CMV promoter and the bovine growth hormone polyadenylation signal to obtain the CMV-RHuT-gD, CMV-RRT-gD and CMV-RRT-Fc expression cassettes. The latter cassettes were excised from the plasmid backbone and sub-cloned into the pINT2 shuttle vector containing two BoHV-4 TK flanking sequences,²⁴ in order to generate the targeting vectors pTK-CMV-RHuT-gD-TK (pINT2-RHuT-gD), pTK-CMV-RRT-gD-TK (pINT2-RRT-gD) and pTK-CMV-RRT-Fc-TK (pINT2-RRT-Fc) (Fig. 1A-C). The resulting constructs were functionally validated in terms of protein expression by transient transfection into HEK 293T cells and immunoblotting with a monoclonal antibody directed against the gD106 tag peptide. All three chimeric proteins were well expressed in transfected cells (Fig. 1D-F) and, as expected, RRT-gD and RRT-Fc were found to be secreted (data not shown).

Figure 1. Design and expression of Her-2 chimeric proteins. Diagrams (not to scale) of (A) pTK-CMV-RHuT-gD-TK (pINT2-RHuT-gD), (B) pTK-CMV-RRT-gD-TK (pINT2-RRT-gD) and (C) pTK-CMV-RRT-Fc-TK (pINT2-RRT-Fc) targeting vectors with expression cassettes under the control of the CMV promoter (pCMV, blue) and the bovine growth hormone polyadenylation signal (PA, orange). RHuT-gD (A) and RRT-gD (B) ORFs are tagged with the gD106 peptide (red), while the RRT-Fc ORF (C) was fused to a mouse IgG Fc encoding fragment (gray). All expression cassettes are flanked by BoHV-4 TK homologous sequences (white). The results of immunoblotting analyses conducted with an anti-gD106 antibody on HEK 293Tcells transfected with pINT2-RHuT-gD, pINT2-RRT-gD and pINT2-RRT-Fc are shown in panels D-F, respectively. Individual lanes were loaded with different amounts of total protein cell extract (5, 10 and 20 μg); cells transfected with pEGFP-1 served as negative controls (Mock).

Immunogenicity profiling of the different HER-2 constructs delivered to syngeneic mice by DNA vaccination

Although all three targeting vectors (pINT2-RHuT-gD, pINT2-RRT-gD and pINT2-RRT-Fc) led to high chimeric Her-2 protein levels in HEK 293T cells, we wished to evaluate their immunogenic properties more directly before converting them to the corresponding viral delivery vectors. To this end, 3 groups of BALB/c mice (6 animals/group) were immunized twice, at 2-weeks intervals, with 50 µg of each plasmid and anti-rat Her-2 humoral and cellular immune responses were evaluated 2 weeks after the second vaccination. A targeting vector, pTK-CMV-A29gD-TK (pINT2-A29-gD), carrying an unrelated antigen from Monkeypoxvirus,³⁶ was administered to a fourth group of mice and served as a negative control. Only mice vaccinated with pINT2-RHuT-gD, the plasmid coding for the membrane-bound form of the antigen, yielded a well-detectable anti-rat-Her-2 antibody response (Fig. 2A). However, as revealed by parallel in vivo cytotoxicity assays using lysis of syngeneic splenocytes pulsed with the immune-dominant (H2^d) rat Her-2 peptide TYVPANASL as readout, all plasmids elicited a specific anti rat-HerR-2 cellular immune response (Fig. 2B). Similarly, all plasmids triggered IFNγ-producing cells upon TYVPANASL peptide re-stimulation in ELISPOT assays (Fig. 2C).

Figure 2. Anti-rat-Her-2 immune responses induced by DNA vaccination with the different pINT2 expression plasmids. (A) Sera from BALB/c mice collected two weeks after the first (gray dots; n=6) and the second (black dots; n=6) vaccination were analyzed (at a dilution of 1:100) for the presence of specific anti-rat-Her-2 antibodies by flow cytometry. Results are expressed as MFI values for each serum. Horizontal lines represent median values (**: p = 0.003, Student's t test). (B) In vivo cytotoxic responses against the H2^d dominant, rat Her-2 TYVPANASL peptide measured two weeks after the second vaccination in mice (n=3) immunized with the indicated pINT2 plasmids; data are mean values ± SEM. (*: p = 0.02; **: p = 0.005; ***: p <0.0001; Student's t-test). (C) T-cell responses against the H2^d dominant, rat Her-2 TYVPANASL peptide measured in vitro, two weeks after the second vaccination, in mice (n=3) immunized with the indicated pINT2 plasmids, using an IFNγ-based ELISPOT assay. Data, expressed as SFU/1×10⁶ SPC, are presented as mean ± SEM values (**: p = 0.001, ***: p = 0.0008; Student's t-test).

To determine immunogenicity in a mouse model more closely resembling the cancer situation (i.e., Her-2 tolerance), we next evaluated the ability of pINT2-RHuT-gD, pINT2-RRT-gD and pINT2-RRT-Fc to induce anti-rat Her-2 antibodies in BALB-neuT mice.³⁷ These mice display a central tolerance with deletion of rat Her-2 TYVPANASL peptide-reactive CD8⁺ T cells.^14,15 Following vaccination (16-week-old BALB-neuT mice; n=7 per group), only animals receiving the pINT2-RHuT-gD plasmid displayed a significant anti-rat Her-2 humoral immune response (Fig. S4). As expected, given the expression of rat-Her-2 in the thymus of newborn BALB-neuT mice,²¹ these animals failed to mount any in vivo cytotoxic response against the TYVPANASL peptide and no IFNγ was produced by splenocytes derived from vaccinated BALB-neuT mice upon TYVPANASL re-stimulation (data not shown).

Despite the unique ability of pINT2-RHuT-gD to elicit anti-rat Her-2 antibodies in both mouse strains, all plasmids appeared to be capable to induce cellular immune responses in BALB/c mice. Therefore all three targeting vectors were carried on and used to construct the corresponding recombinant viruses.

Construction of recombinant BoHV-4 viruses containing different HER-2 expression cassettes

The genome molecular clone of a safe BoHV-4 isolate (designated as BoHV-4-A) derived from the milk cell fraction of a clinically healthy cow²⁶ was used to construct the three recombinant HER-2-BoHV-4 vectors (BoHV-4-RHuT-gD, BoHV-4-RRT-gD and BoHV-4-RRT-Fc) plus a control viral vector (BoHV-4-A29-gD) delivering a completely unrelated antigen. To this end, pINT2-RHuT-gD, pINT2-RRT-gD, pINT2-RRT-Fc and the pINT2-A29-gD plasmid vectors were first linearized by a restriction enzyme digestion sparing the BoHV-4 TK flanking regions. Linearized plasmids were then electroporated into SW102 E. coli cells containing the artificial chromosome pBAC-BoHV-4-A-KanaGalKΔTK^26,38,39 (Fig. 3A), in order to generate pBAC-BoHV-4-A-CMV-RHuT-gD-ΔTK, pBAC-BoHV-4-A-CMV-RRT-gD-ΔTK, pBAC-BoHV-4-A-CMV-RRT-Fc-ΔTK (Fig. 3B) and pBAC-BoHV-4-A-CMV-A29-gD-ΔTK artificial chromosomes via heat-inducible homologous recombination.⁴⁰ The TK locus of the BoHV-4 genome is extremely stable even after repeated passages in vitro and in vivo, and it can thus be reliably employed to integrate foreign DNA sequences into the BoHV-4 genome without any transgene or viral replication efficiency loss due to recombination.^24-30

Figure 3. Recombinant BoHV-4 constructs. (A) Diagram (not to scale) illustrating the re-targeting event (i.e., replacement of the Kana/GalK cassette with the CMV-RHuT-gD, CMV-RRT-gD and CMV-RRT-Fc expression cassettes) generated by heat-inducible homologous recombination in SW102 E. coli cells containing pBAC-BoHV-4-A-TK-KanaGalK-TK. (B) Two representative, 2-deoxy-galactose resistant colonies for each recombinant pBAC-BoHV-4 genome, tested by Hind III restriction enzyme analysis and DNA gel blotting performed with a probe targeting the rat HER-2 portion of each chimeric ORF. The 2,650 bp band (circled in yellow) corresponding to the non-retargeted pBAC-BoHV-4-A-TK-KanaGalK-TK control is replaced by 2,825 bp, 3,642 bp and 3,464 bp bands (circled in red) in pBAC-BoHV-4-RRT-gD, pBAC-BoHV-4-RHuT-gD and pBAC-BoHV-4-RRT-Fc, respectively.

Selected SW102 E. coli clones carrying pBAC-BoHV-4 recombinants were analyzed by HindIII restriction enzyme digestion and confirmed by DNA blotting with probes specific for the three chimeric ORFs (Fig. 3B). Stability of the pBAC-BoHV-4 recombinant clones in E. coli cells (i.e., the absence of restriction pattern alterations upon artificial chromosome propagation) was verified by restriction enzyme digestion after multiple (up to 20) serial passages (Fig. S5).

To produce viable, replication-competent recombinant viral particles, pBAC-BoHV-4-A-CMV-RHuT-gD-ΔTK, pBAC-BoHV-4-A-CMV-RRT-gD-ΔTK and pBAC-BoHV-4-A-CMV-RRT-Fc-ΔTK DNA constructs were electroporated into standard or cre recombinase-expressing BEK cells. The latter cells stably express the phage D1 cre recombinase²⁶ and allow for the site-specific removal of the floxed, GFP cassette-containing BAC sequence from the BAC-BoHV-4 genome. As a consequence of this removal and new cassette insertion, viral plaques generated on a BEKcre cell monolayer lost the characteristic GFP fluorescence compared to parallel plaques seeded onto a standard BEK cell monolayer (Fig. 4A, D and G). Although viable BoHV-4-RHuT-gD, BoHV-4-RRT-gD and BoHV-4-RRT-Fc virus particles were successfully reconstituted in BEK or BEKcre cells, as demonstrated by the cytopathic effect (CPE) observed in the cell monolayer, it was of interest to determine their replication properties with respect to the parental BoHV-4-A virus. As apparent in Fig. 4B, E and H, BoHV-4-RHuT-gD, BoHV-4-RRT-gD and BoHV-4-RRT-Fc, all displayed a slower replication rate compared to the reference BoHV-4-A type. Furthermore, as revealed by immunoblotting analysis of infected cell extracts, they all expressed the corresponding HER-2 transgenes (Fig. 4C, F and I) and as expected, BoHV-4-RHuT-gD targeted transgene expression to the cell membrane (Fig. S6).

Figure 4. Reconstitution and characterization of recombinant viruses. Representative phase contrast and fluorescent microscopy images of the plaques formed by viable, reconstituted recombinant BoHV-4-RHuT-gD (A), BoHV-4-RRT-Fc (D) and BoHV-4-RRT-gD (G) after electroporation of the corresponding BAC DNA clones into BEK or BEKcre cells (magnification, ×10). Replication rates of BoHV-4-RHuT-gD, BoHV-4-RRT-Fc and BoHV-4-RRT-gD grown in BEK cells are shown in panels (B), (E) and (H), respectively, and compared with those of the parental BoHV-4-A isolate. The data mean ± standard error of triplicate measurements (p >0.05 for all time-points; Student's t-test). The results of immunoblotting analyses conducted on extracts from cells infected with BoHV-4-RHuT-gD, BoHV-4-RRT-Fc and BoHV-4-RRT-gD are shown in panels (C), (F) and (I), respectively; BoHV-4-A infected cells served as negative controls.

Higher immunogenicity of HER-2 antigens delivered as BoHV-4 recombinant viral particles compared to DNA immunization

To test the immunogenicity of the different recombinant virus particles, 4 groups of BALB/c mice (7 animals/group) were vaccinated twice intraperitoneally (i.p.), at two weeks intervals, with BoHV-4-RHuT-gD, BoHV-4-RRT-gD and BoHV-4-RRT-Fc, plus the unrelated BoHV-4-A29-gD control. Two weeks after the second immunization, sera were collected and tested for the presence of anti-rat-Her-2 antibodies. No specific anti-rat-Her-2 humoral immune response was detected in mice vaccinated with either BoHV-4-RRT-gD or BoHV-4-RRT-Fc. In contrast, BoHV-4-RHuT-gD viral particles elicited a sustained anti-rat-Her-2 antibody response (Fig. 5A), significantly higher (˜2.5-fold) than the one previously detected in BALB/c mice immunized with the pINT2-RHuT-gD plasmid (p = 0.02) (Fig. 2A). Also, while BoHV-4-A29-gD viral particles did not induce any appreciable cytotoxic response against TYVPANASL-pulsed syngeneic splenocytes, all HER-2 containing recombinant BoHV-4 viral particles induced a strong cytotoxicity (Fig. 5B), significantly higher (˜3-fold) than that induced by the corresponding pINT2 plasmids delivered through DNA vaccination (p = 0.009 for BoHV-4-RHuT-gD vs. pINT2-RHuT-gD; p = 0.03 for BoHV-4-RRT-gD vs. pINT2-RRT-gD; p < 0.0001 for BoHV-4-RRT-Fc vs. pINT2-RRT-Fc). Similarly, IFNγ–producing cells were induced at high frequency by TYVPANASL peptide restimulation of BoHV-4-HER-2- vaccinated mice (Fig. 5C), with no statistically significant difference between the three experimental groups.

Figure 5. Anti-Her-2-specific immune responses induced by intraperitoneal vaccination with the different recombinant BoHV-4 viral particles. (A) Sera from BALB/c mice (n = 7), collected two weeks after the second vaccination, analyzed (at a 1:100 dilution) for the presence of specific anti-rat-Her-2 antibodies by flow cytometry. Results are expressed as MFI values for each serum; horizontal lines represent median values (***: p < 0.0001; Student's t-test). (B) In vivo cytotoxic responses against the H2^d dominant, rat Her-2 peptide TYVPANASL measured in mice immunized with the indicated BoHV-4 viral particles (n = 3) two weeks after the second vaccination; data are mean ± SEM values (***: p = 0.0004; Student's t-test. (C) T-cell responses against the H2^d dominant, rat Her-2 TYVPANASL peptide determined two weeks after the second vaccination by an in vitro IFNγ-based ELISPOT assay. IFNγ-producing cells from mice immunized with the indicated BoHV-4 viral particles (n = 3) are expressed as SFU/1×10⁶ SPC; data are mean ± SEM values (***: p < 0.0001; Student's t-test).

BoHV-4-RHuT-gD affords a significant protection against rat-HER-2-driven mammary carcinogenesis in BALB-neuT mice

In HER-2-tolerant BALB-neuT mice, similarly to what we observed after vaccination with pINT2 plasmid DNA (Fig. S4), only BoHV-4-RHuT-gD (i.e., BoHV-4-A containing the RHuT-gD expression cassette) effectively induced anti-rat-Her-2 antibodies, at levels considerably higher (˜3-folds) than those elicited by pINT2 (Fig. 6A). The superior immunogenicity of BoVH-4-RHuT-gD is also supported by the earlier appearance of anti-rat Her-2 antibodies, which were already well detectable after the first vaccination and increased thereafter, reaching titers significantly higher than those elicited by pINT2-RHuT-gD (p <0.0001). Most importantly, the presence of anti-rat-Her-2 antibodies in sera from BALB-neuT mice vaccinated with BoHV-4-RHuT-gD correlated with a significant delay of mammary tumor appearance. In fact, 50% of BoHV-4-RHuT-gD-vaccinated BALB-neuT mice were completely tumor-free at week 30, when 100% of BoHV-4-RRT-gD- and BoHV-4-RRT-Fc-vaccinated animals already displayed at least one palpable tumor (Fig. 6B).

Figure 6. Anti-rat-Her-2 antibody production and delayed mammary tumor appearance induced by vaccination with BoHV-4-RHuT-gD viral particles. (A) Sera from BALB-neuT mice, collected two weeks after the first (gray dots; n = 8 to 17 animals/group) and the second (black dots; n = 7-17 animals/group) i.p. immunization with the indicated BoHV-4 particles, were analyzed by flow cytometry (at a 1: 100 dilution) for the presence of specific anti-rat-Her-2 antibodies. Results are expressed as MFI values for each serum; horizontal lines represent median values (***: p < 0.0001; Student's t-test). (B) Mammary tumor incidence in control BoHV-4-A29-gD (dotted gray line, n = 8), BoHV-4-RRT-gD, (dotted black line, n = 11), BoHV-4-RRT-Fc (solid black line, n = 11) and BoHV-4-RHuT-gD (solid red line, n = 17) BALB-neuT mice vaccinated with the indicated BoHV-4 viral particles (***: p <0.0001; Mantel–Haenszel Log-rank test).

Discussion

The aim of this study was to investigate the potential of BoHV-4 as a safe, potent and large-capacity vaccine vector able to deliver HER-2-derived engineered antigens and to protect HER-2 transgenic, BALB-neuT mice from autochthonous mammary cancer. BALB-neuT mice were used as a model system because they share a number of features with human breast cancer. In fact, following multi-step progression,¹⁴ each BALB-neuT mammary gland spontaneously develops an independent rat-Her-2⁺ tumor,^12,41 which becomes invasive and metastasizes to the bone marrow first and subsequently to the lungs.⁴² Moreover, similarly to patients with Her-2⁺ carcinomas, BALB-neuT mice lack high-affinity, Her-2 peptide-recognizing cytotoxic T lymphocytes,¹⁵ with an expansion of T regulatory ¹⁴ and myeloid immature suppressor cells⁴³ during carcinogenesis progression. For all these reasons, BALB-neuT mice represent an excellent model system to set up and test novel therapeutic and immune-prophylactic approaches to control breast cancer.³⁷

Initially, CMV-RHuT-gD, CMV-RRT-gD and CMV-RRT-Fc expression cassettes were successfully tested in terms of immune response by DNA vaccination. While all plasmids were able to induce a significant anti-rat Her-2 cellular immune response, only mice vaccinated with pINT-2-RHuT-gD displayed a significant production of anti-rat Her-2 antibodies. As expected and already seen in other rat-HER-2 transgenic (CB6F1-neuT) mice vaccinated with a DNA plasmid coding for RHuT, the antibody titer measured in BALB-neuT mice was significantly lower than that of BALB/c mice, being the former deeply tolerant to rat-Her-2 protein.²¹

The fact that mice vaccinated with the plasmids coding for the secreted forms of rat Her-2 did not produce anti-rat Her-2 antibodies was somehow surprising considering that we have previously demonstrated that vaccination with the soluble extracellular domain (ECD) of the rat Her-2, administered either as DNA or as protein, is able to induce serum anti-rat Her-2 antibodies.^41,44 However, in the present work the Her-2 soluble portion encoded by the pINT-2-RTT-gD and pINT-2-RTT-Fc plasmids contains only the first 390 amino acids of the rat protein. The majority of antibody-recognized epitopes is conformational and is made up of discontinuous segments of a peptide that are brought together during the protein folding process.⁴⁵ In the case of the rat Her-2 protein it has been demonstrated that a stable conformation of the ECD is achieved thanks to a large interface between domains I and III.⁴⁶ It is thus conceivable that the lack of part of domain III (from amino acid 391 to amino acid 488)⁴⁷ in the soluble rat Her-2 protein encoded by our plasmids prevents the classical folding, leading to the exposition of epitopes not expressed or inaccessible in the naïve protein.

We then constructed the three corresponding BoHV-4 vectors and evaluated their replication capacity by comparison with the parental BoHV-4-A strain. HER-2-containing recombinant viruses displayed a slower replication, likely attributable to a toxic effect caused by transgene over-expression. Despite their lower replication rate, however, all recombinant viruses abundantly expressed HER-2-derived transgenes in infected cells and triggered sustained T cell immune responses in BALB/c mice. In contrast, BoHV-4-RHuT-gD only was found to be capable of inducing a strong humoral anti-rat Her-2 response in BALB/c and BALB-neuT mice. In both mouse strains, the intensity of this response was significantly higher than that observed in mice vaccinated with the RHuT-encoding pINT-2 plasmid and an earlier anti-rat Her-2 antibody response, already detectable after the first vaccination, was observed in BALB-neuT mice. This sustained antibody production likely explains the striking delay in mammary cancer appearance brought about by BoHV-4-RHuT-gD vaccination. In fact, anti-rat Her-2 antibodies have previously been shown to cause a marked downregulation and cytoplasmic confinement of rat Her-2 both in vitro^13,41 and in vivo,^12,21 with a concomitant impairment of Her-2 mediated PI3K/Akt signaling.⁴⁸ In addition, anti-Her-2 antibodies may activate complement-mediated lysis and antibody-dependent cytotoxicity.^12,21

BoHV-4-delivered, membrane-bound rat-human Her-2 proved to be superior to the same antigen delivered through DNA vaccination with regard to tolerance breaking and humoral immune response induction. This suggests that BoHV-4-based Her-2 vaccines might be superior to pINT2-based Her2 vaccines also in preventing mammary tumor formation. It should be noted that BoHV-4-RHuT-gD vaccine protects 20% of BALB-neuT mice for all their life, a result never obtained after two immunizations against Her-2 using other vaccination strategies, performed at the same stage of tumor progression as in the present work.^21,49-51 Optimization of dosage, administration route and boosting schedule of BoHV-4-RHuT-gD vaccine, would result in a even more sustained antitumor efficacy. Further experiments are warranted to demonstrate this issue.

Various anti-Her-2 vaccine formulations have been tested in recent years, both in pre-clinical cancer models and in the clinic. These include allogenic Her-2⁺ tumor cells,^52-55 Her-2 peptide-presenting autologous dendritic cells,^55,56 Her-2 protein/peptide immunogens,^57,58 Her-2-based DNA vaccines,^12,21, ^23,41, ⁵⁹ virus-like particles carrying Her-2^50,60 and even a chimeric recombinant Her-2 antigen expressed by an attenuated strain of Listeria monocytogenes.⁶¹ Viral delivery vectors, instead, have received much less attention, recombinant vaccinia^62,63 and adenovirus based vectors are the only viral vectors tested, with quite encouraging results, so far.^51,64 This likely reflects potential concerns with safety and anti-vector immunity. Since risk associated with virus-mediated delivery represents a major issue in viral vector development, attenuation is usually regarded as a highly desirable feature and many efforts are directed toward the development of highly attenuated viral strains with decreased virulence. In this study, we took advantage of the natural non-pathogenicity of BoHV-4, which was previously proved in both standard and genetically modified mouse strains.^28,33 Further to this point, we also previously inoculated high BoHV-4 doses intracerebrally with no apparent negative side-effect, and found that the virus effectively transduced brain cells in the area of inoculation, leading to high-level expression of the GFP transgene.⁶⁵ Therefore, BoHV-4 naturally behaves as a replication-incompetent viral vector that does not require further attenuation. One other major advantages of BoHV-4 is its natural inability to induce serum neutralizing antibody responses. This alleviates most concerns regarding the occurrence of pre-existing, host anti-vector antibodies (as it is the case for adenovirus-based vectors) and allows for multiple immunizations, if required.

In conclusion, our study highlights the favorable properties and potential advantages of BoHV-4 as a highly effective viral vector for cellular Her-2 delivery in order to achieve mammary cancer prophylaxis through a potential one-shot active immunization. Given the previous demonstration of the oncolytic properties of BoHV-4,²⁹ future work will address the feasibility (and efficacy) of combined prophylactic and therapeutic approaches based on the use of this particular viral vector.

Materials and methods

Cell lines

Bovine embryo kidney [(BEK) from Dr. M. Ferrari, Istituto Zooprofilattico Sperimentale, Brescia, Italy; (BS CL^-94)], BEK expressing cre recombinase (BEK cre),²⁶ Human Embryo Kidney 293T [(HEK 293T) ATCC: CRL-11268], Mus musculus mammary gland [(NMuMG) ATCC: CRL-1636] and NIH3T3 murine fibroblasts expressing rat-Her-2 protein (3T3/NKB cells)⁶⁶ cell lines were cultured in 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% CO₂.

PCR

The 2,067 bp rat-human transmembrane protein (RHuT) and the 1,250 bp rat ECD of rat Her-2 protein (RRT) were ampl-ified from pVAX RHuT plasmid⁶⁷ with NheI-RHuT sense (5′-CCCGCTAGCCCACCATGATCATCATGGAGCTGGCG-GCC-3′) and SalI-RHuT antisense (5′-CCCCGAGTCGACCTTCCGGATCTTCTGCTGCCGTCG-3′) and with NheI-RRT sense (5′-CCCGCTAGCCCACCATGATCATCATGGAGCTG-3′) and SalI-RRT antisense (5′-CCCCGAGTCGACTGTGATCTCCTCCAGGGTTTCGAAC-ACTTGGAG3′) primer pairs, respectively.

The PCR amplification reactions were carried out in a final volume of 50 μL, containing 10 mM Tris–hydrochloride pH 8.3, 0.2mM deoxynucleotide triphosphates, 3 mM MgCl2, 50 mM KCl and 0.25 μM of each primer. One hundred nanograms of DNA was amplified over 35 cycles, each cycle consisting of denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min and chain elongation with 1U of Pfu DNA polymerase (Fermentas) at 72°C for 150 sec, in the case of RHuT, and at 72°C for 90 sec, in the case of RRT. The so generated 2,067 bp and 1,250 bp amplicons were then checked in 1% agarose gel and visualized after ethidium bromide staining in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA).

Constructs generation

The NheI-RHut-SalI amplified fragment (2,067 bp) was firstly sub-cloned inside the previously NheI/SalI digested pIgkE2gD106, an eukaryotic expression vector containing a gD106 epitope of Bovine herpesvirus 1 glycoprotein D, successfully used as a tag during the cloning.³⁵ The obtained pIgkRHuTgD106 was subsequently digested with NheI/BamHI to insert the 2,169 bp fragment RHuTgD106 into pEGFP-C1 vector (Addgene), digested with the same enzymes, to remove EGFP gene and to generate pCMVRHuTgD106. Finally, the 2,421 bp Nhe/MluI-blunt ended fragment, containing RHuTgD106 with Simian Virus 40 poly A, was excised and inserted inside pINT2EGFPTK shuttle vector,²⁴ cut with NheI/SmaI restriction enzymes, to obtain pINT2-RHuT-gD.

The amplified NheI-RRT-SalI (1,250 bp) fragment was sub-cloned into NheI/SalI previously digested pIgkE2gD106³⁵ and pIgkE2Fc, an eukaryotic expression vector expressing the crystallizable fragment (Fc) of mouse Immunoglobulin, used as a tag and soluble secreted fragment. RRT-gD106 (1,352 bp) and RRTFc (1,992 bp) were excised with the double digestion NheI/BamHI and inserted into NheI/BamHI digested pEGFP-C1 (Addgene), to remove EGFP gene and generate pCMV-RRT-gD106 and pCMV-RRT-Fc. NheI/MluI-blunt ended fragments containing RRT-gD106 (1,604 bp) or RRT-Fc (2,244 bp), containing the Simian Virus 40 poly A, were excised and inserted inside pINT2EGFPTK shuttle vector,²⁴ cut with NheI/SmaI restriction enzymes, to obtain pINT2-RRT-gD and pINT2-RRT-Fc.

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 pINT2-RHuT-gD, pINT2-RRT-gD and pINT2-RRT-Fc, using Polyethylenimine (PEI) transfection reagent (Polysciences, Inc.). Briefly, 3 μg of DNA were mixed with 7,5 μg PEI (1mg/mL) (ratio 1:2.5 DNA-PEI) in 200 μL of Dulbecco's modified essential medium (DMEM) at high glucose percentage (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 on the cells for 6 h at 37°C with 5% CO₂ in air, in a humidified incubator. The transfection mixture was then replaced with fresh medium (EMEM, with 10% FBS, 50 IU/mL of penicillin, 100 μg/mL of streptomycin and 2.5 μg/mL of Amphotericin B) and incubated for 24 h at 37°C with 5% CO₂.

Viruses

BoHV-4-RHuT-gD, BoHV-4-RRT-gD, BoHV-4-RRT-Fc and BoHV-4-A were propagated by infecting confluent monolayers of BEK cells at a multiplicity of infection (M.O.I.) of 0.5 50% tissue culture infectious doses (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 interested 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.⁶⁵ Virus pellets were then resuspended in cold EMEM without FBS. TCID₅₀ were determined with BEK cells by limiting dilution.

Western immunoblotting

Protein cell extracts were obtained from a six-well confluent plate of HEK 293T transfected with pINT2-RHuT-gD, pINT2-RRT-gD and pINT2-RRT-Fc and from 25-cm² confluent flasks of BEK infected with BoHV-4-RHuT-gD, BoHV-4-RRT-gD, BoHV-4-RRT-Fc 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), diluted 1:15.000, and then with a secondary antibody probed with horseradish peroxidase-labeled anti-mouse immunoglobulin (A 9044; Sigma), diluted 1:10.000, to be visualized by enhanced chemiluminescence (ECL Kit; Pierce). pINT2-RRT-Fc and BoHV-4-RRT-Fc protein extracts were directly incubated with the secondary antibody probed with horseradish peroxidase-labeled anti-mouse immunoglobulin (A 9044; Sigma), recognizing the Fc tag.

BAC recombineering and selection

Recombineering was performed as previously described⁴⁰ with some modifications. Five hundred microliters of a 32°C overnight culture of SW102 containing BAC-BoHV-4-A-Kana-GalKΔTK, were diluted in 25 ml Luria–Bertani (LB) medium with or without chloramphenicol (SIGMA) selection (12.5 mg/mL) in a 50 mL baffled conical flask and grown at 32°C in a shaking water bath to an OD₆₀₀ of 0.6. Then, 10 mL were transferred to another baffled 50 mL conical flask and heat-shocked at 42°C for exactly 15 min in a shaking water bath. The remaining culture was left at 32°C as the un-induced control. After 15 min the two samples, induced and un-induced, were briefly cooled in ice/water bath slurry and then transferred to two 15 mL Falcon tubes and pelleted using 5,000 r.p.m. (eppendorf centrifuge) at 0°C for 5 min. The supernatant was poured off and the pellet was resuspended in 1mL ice-cold ddH₂O by gently swirling the tubes in ice/water bath slurry. Subsequently, 9 mL ice-cold ddH₂O were added and the samples pelleted again. This step was repeated once more, the supernatant was removed and the pellet (50 μL each) was kept on ice until electroporated with gel-purified PvuI (Fermentas) linearized pINT2-RHuT-gD, pINT2-RRT-gD and pINT2-RRT-Fc. An aliquot of 25 μL was used for each electroporation in a 0.1 cm cuvette at 25 μF, 2.5 kV and 201Ω. After electroporation, for the counter selection step, the bacteria were recovered in 10 mL LB in a 50 mL baffled conical flask and incubated for 4.5 h in a 32°C shaking water bath. Bacteria serial dilutions were plated on M63 minimal medium plates containing 15 g/L agar, 0.2% glycerol, 1mg/L D-biotin, 45 mg/L L-leucine, 0.2% 2- deoxy-galactose and 12.5 mg/mL chloramphenicol. All the complements for M63 medium were purchased from SIGMA.

Plates were incubated 3–5 d at 32°C; then several selected colonies were picked up, streaked on McConkey agar indicator plates (DIFCO, BD Biosciences) containing 12.5 mg/mL of chloramphenicol and incubated at 32°C for 3 d until white colonies appeared. White colonies were grown in duplicate for 5–8 h in 1mL of LB containing 50 mg/mL of kanamycin (SIGMA) or LB containing 12.5 mg/mL of chloramphenicol. 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 overnight in a 1% agarose gel, stained with ethidium bromide, and visualized to UV light.

Original detailed protocols for recombineering can also be found at the recombineering website (http://recombineering.ncifcrf).

Non isotopic 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.²⁶

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 1,250 bp amplicon for RRT digoxigenin-labeled probe was generated by PCR with the primers NheI-RRT sense and SalI-RRT antisense, as previously described.²⁷ PCR amplification was carried out in a final volume of 50 μL of 10 mM Tris–HCl, pH 8.3, containing 0.2 mM deoxynucleotide triphosphates, 0.02 mM alkaline labile digoxigenin-dUTP (Roche), 3 mM MgCl₂, 50 mM KCl, and 0.25 μM of each primer over 35 cycles, each cycle consisting of denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min, and chain elongation with 1 U of Taq polymerase (Thermo Scientific) at 72°C for 90 sec. A parallel reaction omitting digoxigenin dUTP was performed, because digoxigenin incorporation into the amplicon can be checked through the size shift of the amplicon by gel electrophoresis. Five microliters of the probe were added to 500 μL of ddH₂O into a screw-cap tube, denatured in boiling water for 5 min, and cooled down on ice for another 2 min. Denatured probe was added to 50 mL of pre-heated 65°C hybridization solution (7% SDS, 0.5 M phosphate, pH 7.2 and 1 mM EDTA) to the pre-hybridized membrane and hybridized overnight at 65°C in a rotating hybridization oven (Techna Instruments). Following hybridization, the membrane was washed twice for 30 min with 100 mL of washing solution I (0.5 × SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate] and 0.1% SDS) and twice for 30 min with 100 mL of washing solution II (40 mM phosphate, pH 7.2, 0.05% SDS) at 65°C. On a freshly washed dish, the membrane was incubated for 30 min at room temperature in 100 mL of blocking solution (100 mM maleic acid, pH 7.5, 150 mM NaCl, 1% blocking reagent [Roche]). Anti-digoxigenin Fab fragment (150 U/200 μL [Roche]), diluted 1:15.000 in 50 mL of blocking solution, was applied to the membrane for 30 min under gentle shaking at room temperature and washed twice for 15 min with 100 mL of washing solution (100 mM maleic acid, pH 7.5, 150 mM NaCl, 0.3% Tween 20). Detection was performed following equilibration of the membrane in detection buffer (100 mM Tris–HCl, pH 9.5, 1 mM EDTA) for 2 min at room temperature. Chemiluminescent substrate (CSPD, Roche) was added by scattering the drops over the surface of the membrane after placement of the membrane between two plastic sheets, and any bubbles present under the sheet were eliminated with a damp lab tissue to create a liquid seal around the membrane. Signal detection was obtained, exposing the membrane to X-ray film. The exposure time was adjusted with the intensity of the signal.

Cell culture electroporation and recombinant virus reconstitution

BEK or BEKcre cells were maintained as a monolayer with complete EMEM 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 vessel (i.e., every 3–5 d) 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/or BEKcre 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 d 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 per cell and maintaining them in MEM with 10% FBS for 2 h.

Viral growth curves

BEK cells were infected with BoHV-4-A, BoHV-4-RHuT-gD, BoHV-4-RRT-gD, BoHV-4-RRT-Fc 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 µg/mL streptomycin and 2.5 µg/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.

Mice

BALB/c (Charles River) and BALB-neuT (Ariano Irpino, Italy)⁶⁸ mice were bred under specific pathogen-free conditions (Allentown Caging Equipment, Allentown, NJ, USA) at the Molecular Biotechnology Centre (Torino, Italy) and treated according to the European Guidelines and policies, as approved by the University of Torino Ethical Committee. To assess mammary tumor incidence BALB-neuT females were inspected weekly by palpation, and progressively growing masses with a mean diameter of >1 mm were regarded as tumors. Each tumor mass was measured with a calliper in the two perpendicular diameters. Growth was monitored until all 10 mammary glands displayed a tumor or until a tumor exceeded a mean diameter of 10 mm, at which time mice were sacrificed for humane reasons.

Mice immunization

Recombinant pINT2 plasmids were purified by large scale preparation using the EndoFree Plasmid Giga kits (Qiagen, Inc., CA, USA). Ten week-old BALB/c and BALB-neuT mice were anesthetized by intramuscular injection (i.m.) of 40 μL of a solution containing 5.7 μL of Zoletil 100 (Vibrac, Milano, Italia), 3.5 μL of Rompum (Bayer, Milano, Italia) and 37.5 μL of Phosphate Buffer Saline (PBS) (GIBCO, Grand Island, NY, USA). Anesthetized mice were injected in the quadriceps muscle with 50 μg of plasmid DNA diluted in 20 μL of saline solution. Immediately after injection, two 25-ms trans-cutaneous electric low voltage pulses with amplitude of 150 V and a 300 ms interval were administered at the injection site via a multiple needle electrode connected to the Cliniporator™ (IGEA s.r.l., Carpi, Italy). The DNA vaccination course consisted of two i.m. injections of plasmid followed by electroporation repeated with an interval of 14 d.

Ten⁶ TCID₅₀ recombinant BoHV-4 viral particles were diluted in 200 μL of Dubecco's Modified Eagle Medium (DMEM; Gibco, Rockville, MD) and injected intraperitoneal (i.p.) twice at two weeks interval in groups of 10 week-old BALB/c or BALB-neuT females.

Anti-rat-Her-2 antibody response

Two weeks after the second immunization, mice were bled and their sera were tested by flow cytometry for their ability to bind 3T3/NKB cells.⁶⁶ Briefly, sera diluted 1:100 in PBS were incubated for 30 min at 4°C with 2 × 10⁵ 3T3/NKB cells pre-treated with Fc receptor blocker (CD16/CD32; PharMingen, St. Diego, CA) for 5 min at 4°C. The Ab4 mAb (Calbiochem, San Diego, CA), was used as positive control for rat Her-2 positivity. After washes with PBS containing 2% bovine serum albumin (BSA, Sigma-Aldrich, Milano, Italy) and 0.1% NaN3 (Sigma-Aldrich) (wash solution) cells were incubated with 1:50 dilution of a FITC-conjugate anti-mouse immunoglobulin G (IgG) Fc antibody (DakoCytomation, Milano, Italy) for 30 min at 4°C. Washed cells were then acquired and analyzed on the CyAn ADP using Summit 4.3 software (DakoCytomation, Heverlee, Belgium). The results were expressed as Mean Fluorescence Intensity (MFI).

Anti-rat-Her-2 cellular immune response

To prepare target cells for in vivo cytotoxicity detection, spleens from BALB/c and BALB-neuT mice were mechanically dissociated and the erythrocytes were removed from the cells suspension by osmotic lysis. Cells were then washed and labeled with two different CFSE (carboxyfluorescein-diacetate-succinimidyl ester) (Molecular Probes Invitrogen, Carlsbad, CA) concentration (5 and 0.5 μM). Cells labeled with 5 μM CFSE (CFSE_high cells) were also pulsed with the rat-Her-2 H2^d dominant TYVPANASL peptide (INBIOS Srl) at a concentration of 15 μg/mL for 1 h at 37°C; those labeled with 0.5 μM CFSE (CFSE_low cells) were left unpulsed. 10 × 10⁶ CFSE_high cells plus 10 × 10⁶ CFSE_low cells were injected in the tail vein of vaccinated mice. Forty eight hours after spleen cells injection, mice were sacrificed and the presence of CFSE_high and CFSE_low in the spleen was measured by using a CyAn ADP Flow Cytometer (DakoCytomation). The percentage of the low peaks was normalized on control untreated low peaks and consequently the specific cytolytic activity was calculated as percentage of lysis as previously described.⁶⁹

To measure the number of rat-Her-2 specific IFNγ releasing T lymphocytes a mouse IFNγ ELISPOT assay kit purchased from BD Biosciences (San Jose, CA, USA) was used. Briefly, two weeks after the vaccination course, 0.5 × 10⁶ spleen cells were added to the wells of 96-well HTS IP plates (Millipore, Billerica, MA) pre-coated with 5 μg/mL of rat anti-mouse IFNγ (clone R4–6A2, BD Biosciences, San Jose, CA, USA). Spleen cells were stimulated with 15 μg/mL of TYVPANASL peptide (INBIOS Srl, Napoli, Italy) for 24 h at 37°C in a humidified 5% CO2 atmosphere. Concanavalin A (Sigma-Aldrich) at the concentration of 2 µg/mL and RPMI-1640 medium (Sigma-Aldrich) alone were used as positive and negative control, respectively. IFNγ spots were scanned and counted using an ImmunoSpot Image Analyzer software (Aelvis, Germany). Results were plotted as median of spot values among triplicates.

Immunofluorescence assay

For rat Her-2 detection, 4 × 10⁵ Nmug cells were plated on glass coverslips and left to adhere overnight at 37 °C in a 5% CO₂ incubator. The next day, cells were infected for 24 h with 0.5 TCID₅₀/cell of BoHV-4-RHuT-gD or with the same TCID₅₀/cell of BoHV-4-RHuT-gD inactivated at 70°C for 30 min. After infection, cells were fixed with 4% formalin (Sigma-Aldrich) solution in PBS for 5 min at room temperature, washed twice with PBS and blocked with 10% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 40 min at room temperature. Her-2 was detected incubating coverslips with an anti rat/Her-2 monoclonal antibody (1:20, clone number, Calbiochem, San Diego, CA) for 1 h at room temperature in PBS containing 1% BSA. Cells were rinsed twice with PBS and then incubated with AlexaFluor488 goat anti-mouse (1:1000, clone A11017, Invitrogen) in PBS containing 1% BSA. Cells were rinsed three times with PBS and nuclei were counterstained with DAPI (Invitrogen). Coverslips were air dried and mounted with Fluoromount mounting medium (Sigma-Aldrich) and visualized with Apotome fluorescence microscope (Leica). Photographs were taken using a digital CCD camera and images were processed using the AxioVision software (Zeiss, V. 4.4).

FACS analysis

For rat Her-2 detection, 4 × 10⁵ Nmug cells were plated and left to adhere overnight at 37°C in a 5% CO₂ incubator. The next day, cells were infected for 24 h with 0.5 TCID₅₀/cell of BoHV-4-RHuT-gD or with the same TCID₅₀/cell of BoHV-4-RHuT-gD inactivated at 70°C for 30 min. After infection, cells were detached with tripsin 1X (Invitrogen o Sigma), incubated with Fc receptor blocker (CD16/CD32; PharMingen, St. Diego, CA) for 5 min at 4°C to block aspecific site. Her-2 was detected incubating cells with an anti rat/Her-2 monoclonal antibody (1:25, clone number, Calbiochem, San Diego, CA) for 30 min at 4°C. Then cells were washed with PBS containing 2% BSA (Sigma-Aldrich, Milano, Italy) and 0.1% NaN3 (Sigma-Aldrich) (wash solution) and incubated with 1:50 dilution of a FITC-conjugate anti-mouse immunoglobulin G (IgG) Fc antibody (DakoCytomation, Milano, Italy) for 30 min at 4°C. Washed cells were then acquired and analyzed on the CyAn ADP using Summit 4.3 software (DakoCytomation, Heverlee, Belgium).

Statistical analysis

Statistical differences were evaluated using GraphPad software 5.0 (GraphPad Inc..). The Mantel-Cox log-rank test was used to evaluate the differences in the tumor incidence between different experimental groups. The two-tailed unpaired Student's t test was used to evaluate differences in the antibody titer, % of lysis and number of IFNγ secreting T cells between different experimental groups.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed

Acknowledgments

This work was supported by grants from the Italian Association for Cancer Research (AIRC, grant IG 12956 and IG 11675), the Compagnia di San Paolo (Progetti di Ricerca Ateneo/CSP, TO_call02_2012_0026), the University of Torino (Italy), the Italian Ministry for Education, University and Research (MIUR), Grant # 2010LLXR94_004 and University of Parma (Italy).