Background
The hallmark of poxviruses utilization in anti-cancer immunotherapy is their ability to express large foreign genes without significant disruption of the viral genome. This feature offers the possibility of expressing complex eukaryotic sequences or multiple genes in permissive mammalian cells, ensuring correct post-translational modifications [
1]. To date, different poxviridae genera have been successfully used as tumor associated antigens vectors in experimental models. Engineered attenuated recombinant vaccinia virus has now been widely employed as a cancer vaccine in a large number of clinical trials as well. The results of these trials demonstrated that recombinant vaccinia virus infection upon vaccination was safe and that a specific humoral or T cell response against the foreign inserted tumor-associated antigen could be induced in several cancer patients [
2‐
13]. Vaccination with recombinant vaccinia virus can be achieved by systemic (subcutaneous, intradermal, or intramuscular) or intratumoral injection [
2,
4‐
18]. Recently, it was demonstrated that the antitumor activity induced by intratumoral vaccination with an avipox virus expressing carcinoembryonic antigen (CEA) and multiple co-stimulatory molecules was superior to that induced by subcutaneous vaccination in CEA-transgenic mice [
19]. Similarly, we reported that the intramammary gland vaccination with the recombinant vaccinia virus
neu (rV-
neu T) vaccine was more effective than the subcutaneous vaccination in inhibiting mammary carcinogenesis in BALB-
neu T mice [
20]. In addition, the intramammary delivery was more effective than the subcutaneous vaccination in eliciting anti-Neu antibodies, increasing anti-Neu IgG2a/G3 isotypes and inducing antibodies able to trigger mammary tumor cells apoptosis and antibody-dependent cellular cytotoxicity [
20]. A prerequisite to using intratumoral delivery is the easy access for antigen delivery to the tumor site. Salivary gland tumors as well as head and neck tumors including tongue, floor of the mouth, palate and mandibular mucosa and so on, appear suitable for such vaccine delivery. Salivary gland tumors are a group of heterogeneous lesions which express ErbB2, whose current treatment involves surgery and adjuvant radio(chemo)therapy. However, therapy response rates have been generally poor for these tumors [
21]. Recently, given that the high histopatological similarity between salivary ductal and breast carcinomas, Trastuzumab, a humanized monoclonal antibody to ErbB2, has been proposed as a potential therapy for salivary gland tumors treatment. However, active immunization targeting ErbB2 might induce tumor growth inhibition more efficiently than passive immunotherapy based on the generation of an extended memory immune response.
In this study we examined the effectiveness of the rV-neu T intratumoral vaccination in hampering the growth of transplanted Neu-overexpressing BALB-neu T salivary gland cancer cells (H-2d) (SALTO) in BALB-neu T mice. In addition, we explored whether the efficiency of vaccination was dependent on the dose of the rV-neu T vaccine (108 vs 107 vs 106 pfu). Considering previous demonstration that a potent anti-Neu humoral response is required to prevent mammary tumor growth in BALB-neu T vaccinated mice, we investigated the anti-Neu humoral response following rV-neu T vaccination as well as the in vitro biological activity of immune sera from rV-neu T vaccinated mice. Finally, we determined whether rV-neu T vaccination elicits anti-Neu T cell immunity.
Our research suggests that intratumoral vaccination using recombinant vaccinia virus could be efficiently employed for the treatment of salivary gland tumors and other accessible tumors.
Discussion
The incidence of head and neck carcinomas (HNC) is increasing worldwide and despite advances in their treatment, the survival rate of patients with this type of cancer has not substantially changed over the last two decades [
43]. Salivary gland carcinomas are head and neck tumors of heterogeneous morphology that require typical surgical and adjuvant therapy [
44]. Conservative surgery with nerve monitoring remains the state-of-the-art. Adjuvant radio(chemo)therapy is shown to increase local tumor control, but overall survival is not automatically enhanced [
44]. Thus, the development of novel therapies can supplement the pharmaceutical armamentarium presently used for the treatment of HNC and salivary gland carcinomas. A significant tumor specific overexpression of all four ErbB receptors including EGFR, ErbB2, ErbB3, and ErbB4 has been reported in head and neck squamous cell carcinomas (HNSCC) [
45,
46]. ErbB2 overexpression was observed in about 20% of patients with salivary duct cancer (SDC) [
47], a rare high-grade aggressive tumor subtype of salivary gland carcinoma. In agreement with both EGFR and ErbB2 overexpression, cetuximab and trastuzumab, which target EGFR and ErbB2, respectively, represent important tools for treatment of salivary gland carcinomas [
48]. Indeed, it was reported that a patient with SDC positive for ErbB2 had a complete objective response after combined treatment with paclitaxel, carboplatin, and trastuzumab [
49]. Similarly, it was described a case of ErbB2-positive metastatic submandibular SDC with a complete and durable clinical response after treatment with trastuzumab in combination with chemotherapy [
50]. In addition, resolution of measurable and minimal residual disease in a patient with salivary duct cancer treated with trastuzumab, lapatinib, and bevacizumab, with treatment ongoing for more than 2 years was observed [
51]. Thirteen patients with SDC and ErbB2 expression were treated with trastuzumab in adjuvant (n = 8) or palliative (n = 5) setting. It was reported that all patients with metastatic disease (5/5 patients) responded to treatment with trastuzumab. One patient achieved a complete response and remains with no evidence of disease 52 months after initiation of trastuzumab. The median duration of response was 18 months [
52]. However, it was also reported in breast cancer patients that the objective response to trastuzumab monotherapy had a median duration of 9 months, and that the majority of responsive patients displayed resistance within 1 year [
53]. Conversely, combination therapy with trastuzumab and an ErbB2/Neu T helper peptide vaccine was well tolerated and it was associated with minimal toxicity in patients with metastatic breast cancer. In addition, the combinatorial approach of the vaccine with passive immunotherapy resulted in prolonged, robust, antigen-specific immune responses in treated patients and induced epitope spreading [
54]. In agreement with these evidences it is reasonable to investigate ErbB2 cancer vaccine approaches with the aim to improve the objective tumor inhibitory response in salivary gland carcinomas.
Poxvirus represents an attractive delivery vehicle of tumor antigens due to the normal post-translational modification of the inserted antigen and strong immunogenicity [
2,
3,
5]. Engineered attenuated recombinant vaccinia virus encoding for tumor associated antigens has now been widely employed as a cancer vaccine in several clinical trials [
4‐
9,
11,
13,
16,
18,
55]. Vaccination with recombinant vaccinia virus can be achieved by systemic or local intratumoral (i.t.) injection [
2,
4‐
9,
11,
13,
16,
18].
Recently, it was demonstrated that the antitumor activity induced by i.t. vaccination with an avipox virus expressing carcinoembryonic antigen (CEA) and multiple co-stimulatory molecules was superior to that induced by systemic (subcutaneous) vaccination in CEA-transgenic mice [
19]. Similarly, we recently demonstrated that local delivery of recombinant vaccinia virus encoding for
neu (rV-
neu T) was superior to systemic vaccination in inhibiting the neu oncogene-mediated mammary carcinogenesis [
20]. Besides, i.t. injection of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine has been reported to significantly inhibit Her-2/neu-expressing tumor growth. The vaccine elicited transformation of immunosuppressive myeloid-derived suppressor cells into TNF-α-secreting neutrophils and reduced the generation of Treg cells [
56]. Similarly, i.t adenovirus (Ad) vaccination supported the generation of both Neu- and Ad-specific T effector cells [
57]. Of note, it was reported that i.t. vaccination with vaccinia-expressing the tumor antigen HY and granulocyte macrophage colony-stimulating factor was able to overcome immunological ignorance to the tumor antigen [
58]. Head and neck cancers are loco-regional diseases that appear at or close to the body surface and are easily accessible. Thus, the accessibility of salivary gland tumors allow one to envision intratumoral immunotherapy in a neoadjuvant setting.
The attempt to use intratumoral vaccination in HNC was reported by Dasgupta
et al. [
59]. In this study it was demonstrated that recombinant vaccinia virus expressing interleukin-2 invoked anti-tumor cellular immunity in an orthotopic murine model of HNC. However, no antigen was delivered by using this approach.
In this report, we investigated the efficacy of the rV-
neu T intratumoral vaccination in hampering the growth of transplanted Neu-overexpressing salivary gland cancer cells (SALTO) in syngeneic, Neu-tolerant BALB-
neu T mice. In addition, we determined whether the efficiency of vaccination was dependent on rV-
neu T doses (10
8-10
6 pfu). Mice transgenic for the rat
neu oncogene (BALB-
neu T) are usually employed to evaluate the ability of ErbB2/Neu vaccines to inhibit the progression of
neu-driven carcinogenesis [
60]. Our observations indicated that the efficiency of vaccination was dose-dependent. Mice vaccinated with 10
8 pfu rV-
neu T had a mean survival time of 27 weeks while those receiving the 10
7 pfu and 10
6 pfu rV-
neu T doses had a mean survival time of 5.25 and 9.33 weeks, respectively. rV-
neu T vaccination at the dose of 10
8 pfu induced regression of transplanted tumors while that at 10
6 e and 10
7 pfu provoked a delay in the tumor growth as compared to V-wt vaccination. The risk of developing tumors in the 10
6 pfu and 10
7 pfu rV-
neu T vaccinated groups was 10.26 and 14.05 in comparison to the 10
8 pfu rV-
neu T vaccinated group. Overall, the mean survival time of mice vaccinated with rV-
neu T, independently of the dose, was 14.8 weeks while of those receiving the V-wt was 2.63 weeks. It is of note that 8/9 rV-
neu T vaccinated mice were tumor free six weeks after the first vaccination and remained in this status until the 30
th week. Conversely, V-wt vaccinated mice were sacrificed for exceeded tumor volume or spontaneously died at the third week after the first vaccination.
We previously established that immune response and antitumor activity were increased by repeated rV-neu T vaccinations. Accordingly, we performed two immunizations. One of the potential drawbacks in the use of many recombinant vaccinia immunizations in patients is that pre-existing and/or stimulated antibody and T cell response to vaccinia virus will preclude the spread of the administered vaccinia virus and thus decrease the expression of the inserted antigen. On the other hand, it should be noted that smallpox was eradicated worldwide more than 25 years ago; thus, young people are no longer vaccinated. In addition, recombinant avipox virus, which has a limited viral replication, can be employed to boost immune response after priming with recombinant vaccinia.
The extent of tumor growth interference in vivo was associated with high serum levels of anti-Neu antibodies, which were able to recognize p185 Neu expressed on SALTO tumor cells. 108 pfu rV-neu T vaccinated mice developed a significantly higher titer of anti-Neu antibodies than 107 and 106 pfu rV-neu T vaccinated mice. Thus, the amount of produced anti-Neu antibodies was coincident with the efficiency of in vivo anti-tumor activity of rV-neu T vaccinated mice.
Individual mechanisms including ADCC, CDC (complement dependent cytotoxicity), induction of apoptosis, or receptor down regulation have been implicated to elucidate the inhibitory effect of anti-ErbB2/Neu antibodies on the growth of cancer cells expressing ErbB2/Neu [
35,
61‐
67]. In this study, we demonstrated that Igs from rV-
neu T vaccinated mice inhibited
in vitro cell proliferation, mediated ADCC and induced apoptosis of SALTO tumor cells. Indeed, immune sera from rV-
neu T vaccinated mice were able to mediate ADCC
in vitr o. Igs of the IgG2a isotype have been shown to mediate a more potent ADCC than other Ig isotypes in mice [
68]. Anti-Neu antibodies of the IgG2a isotype are well represented in sera of rV-
neu T vaccinated mice. Purified Igs from rV-
neu T vaccinated mice were also able to induce inhibition of SALTO tumor cell growth.
Trastuzumab was shown to induce down regulation of p185 Neu receptor and to block receptor function [
42]. We demonstrated that chronic treatment with purified rV-
neu T Igs were able to induce down regulation of p185 Neu receptor in SALTO cells. This biological effect can make the receptor unavailable for ligands binding thus blocking its signal transduction as we observed by revealing inhibition of the MAP kinases cascade upon rV-
neu T Igs incubation of SALTO cells. Moreover, rV-
neu T vaccinated mice purified Igs were able to induce apoptosis of BALB-
neu T tumor cells
in vitro.
It has been demonstrated that cytokines and antibody production are mostly responsible for inhibition of tumor growth in BALB-
neu T mice, while cytotoxic T lymphocytes might have a marginal role [
35,
69]. Here, we found that spleen T cells of rV-
neu T vaccinated mice released IFN-γ and IL-2 upon stimulation with several Neu-specific peptides. Recognition of these epitopes
in vivo potentially activates T cells to secrete IFN-γ thus determining ischemic necrosis at the tumor site. Such immunodominant epitopes might boost an immune response in BALB-
neu T mice. Overall, our study suggests that rV-
neu T i.t vaccination could be employed to induce an efficient antitumor response and reject transplanted salivary gland tumors. A Phase I study of i.t vaccine administration in men with locally recurrent or progressive prostate cancer was performed [
70]. The intraprostatic administration of PSA-TRICOM [encoding transgenes for prostate-specific antigen (PSA) and 3 costimulatory molecules] poxviral vaccine was safe and feasible and could generate a significant immunologic response. Indeed, improved serum PSA kinetics and intense post-vaccination inflammatory infiltrates were seen in the majority of patients after vaccination [
70]. Local vaccination with recombinant vaccinia virus might provide danger signals which can induce a specific immune response by alerting and activating specialized antigen presenting cells expressing costimulatory molecules and thus promoting T and B cell activation [
71]. Active immunization targeting ErbB2 might block tumor growth more proficiently than passive immunotherapy thanks to the activation of a persistent memory immune response. It would also be useful in boosting a spontaneous occurring ErbB2 immune response. Moreover, an ErbB2 vaccine-based therapy might be helpful to a single anti-ErbB2 Mab therapy by concurrently inducing T and B cell immunity to several immunodominant epitopes.
Our findings may have a significant role for planning cancer vaccine protocols for the treatment of salivary gland tumors and other accessible tumors using i.t injection of recombinant vaccinia virus.
Authors’ contributions
LM performed ADCC, cell proliferation, indirect immunofluorescence, immunoprecipitation and analyzed all of the results. MF carried out the statistical analysis and PCR and T cell response. MB followed the mating of mice. PS performed western blotting and analysis of apoptosis. MGG, IT, PL participated in analysis of experiments and results. FL, FC, PN, JS, AM critically revised the manuscript. RB conducted the antitumor animal experiments, carried out experimental design, supervised the project and wrote the manuscript. All authors read and approved the final manuscript.