Background
Prostate cancer remains a major health issue in the present era, largely due to limitation of therapeutic options especially in advanced disease. Prostate cancer represents the most common non-cutaneous cancer and is the second leading cause of cancer related deaths among American men [
1]. There are continuing efforts to discover new treatments for prostate cancer, in particular for advanced disease. Novel therapeutic strategies are needed to prevent progression from localised to advanced disease and to further improve survival outcomes in patients with metastatic disease. Manipulation of the immune system and destruction of cancer cells by the immune activated mechanisms have shown promising results in the treatment of malignant diseases [
2].
Healthy individuals are known to have some immune inhibitory effects on prostate cancer growth (at least early phase of the disease), while progressive tumour development is a result of tumour escape from the immune system. Many factors are involved in tumour immune escape. Blades et
al. [
3] have shown the reduction of MHC-1 expression in 34% of primary prostate cancer and 80% tumours associated with lymph node metastases. Other causes include secretion of inhibitory substances e.g. IL-10, TGF-β [
4], abnormal T-lymphocyte signal transduction [
5] and expression of Fas ligand, which may enable tumour cells to induce apoptosis in Fas expressing tumour infiltrating lymphocytes [
6]. Immunological therapies may overcome these escape pathways and can potentially play an effective role in the management of prostate cancer in isolation or in conjunction with available therapies. Patients with advanced prostate cancer are known to have defective cell mediated immunity [
7]. Both antibody and CD8
+ T-cell immune responses have been reported in patients with advanced prostate cancer [
8‐
10].
For malignant diseases different approaches of active immunisation have been explored, including vaccination with cDNA [
11], RNA [
12], proteins or peptides [
13]. Over the past years, several prostate cancer associated antigens have been reported including prostate specific antigen (PSA), prostate-specific membrane antigen (PSMA) [
14], prostate stem cell antigen (PSCA) [
15] and six transmembrane epithelial antigen (STEAP) [
16]. We have previously demonstrated the potential for electroporation (EP) mediated DNA vaccination with PSCA [
17]. In the present study, we focus on optimisation of
in vivo DNA plasmid vaccination, in terms of dose schedule and combination with CpG oligonucleotides. We investigated the utilisation of a human PSA expressing plasmid in a murine model of prostate cancer. PSA, a serine protease secreted by both normal and transformed epithelial cells, is almost exclusively expressed on prostatic epithelial cells, and its expression is conserved in nearly all advanced prostate cancer [
18]. PSA is widely used as marker for diagnosis and staging of prostate cancer [
19]. Although PSA is a secreted protease, MHC related epitope processing in target PSA expressing cells has been shown to make PSA a valid target for vaccination [
20]. Additionally, a DNA vaccination with plasmid encoding PSA has a potential to evoke specific anti-tumour cellular immune responses [
21].
DNA vaccines induce immune responses by direct expression of the antigen by the host cells. Electric pulse parameters optimal for the plasmid delivery have been shown to enhance humoral immune responses [
22]. Moreover, plasmid DNA contains CpG motifs, which are immune-stimulatory and have been shown to induce potent immunological adjuvant effects [
23,
24]. While gene based vaccines for prostate cancer have been studied previously, optimal vaccine schedule with EP driven plasmid delivery has not been evaluated. This study aims to test various EP vaccination regimens for prostate cancer in an animal model.
Discussion
Electroporation driven immunisation with prostate antigen (hPSA) encoding plasmid resulted in specific broad immune responses with effective tumour containment. For a cancer vaccine, the prevention of tumour progression may be dependent on both humoral and cellular immunity. We have shown that EP mediated DNA delivery is capable of stimulating both arms of the immune system. Both humoral and cell mediated immune responses were observed as indicated by the anti hPSA antibodies production and
in vitro/
in vivo cytotoxicity respectively. These immune responses were antigen specific as in our tumour rechallenge experiments, tumour protection was only observed in mice challenged with transfected cells. In this study, all three tested regimens provided variable effects on tumour growth but repeated vaccination four times on weekly interval resulted more effective immune responses. All mice developed tumours indicating that these regimens did not provide complete tumour protection. Nevertheless, low tumour burden and prolonged survival in immunised mice was achieved with phPSA vaccination with all vaccination regimens. Furthermore, on co-administration of an immune adjuvant, synthetic CpG containing oligonnucleotides, complete tumour protection was achieved in 54% of animals. Immune effects of CpG DNA in infection have been well documented. It has been observed that the release of unmethylated CpG DNA (which is unique to prokaryotes) during an infection provides a 'danger signal' to the innate immune system, triggering a protective immune response that improves the ability of the host to eliminate infecting microbes [
31]. This initiates a cascade of events that culminates in the indirect maturation, differentiation, and proliferation of T cells and natural killer cells [
32]. Together, these cells secrete cytokines and chemokines that create a pro-inflammatory (IL1, IL 6, IL18 and TNFα) and Th1-polarised (IFNγ, and IL 12) immune environment [
33]. These events further facilitate the development of antigen-specific CTLs [
34,
35]. The induction of these immune responses by oligo CpG has encouraged the idea of a potential role of oligo CpG as vaccine adjuvant. In this study, the CpG adjuvant potentiated the specific anti- tumour immunity as observed by complete tumour growth inhibition.
Developments in tumour vaccines are influenced by the substantial success of the various types of vaccines for infectious diseases. The majority of these vaccines for infectious diseases have effective prophylactic roles with limited utility in therapeutic settings. Tumour vaccine studies have clearly shown that vaccines elicit effective responses against early, microscopic tumours, but are ineffective against established, large tumour masses [
36,
37]. These observations led to the idea of generation of prophylactic, rather than therapeutic, cancer vaccines [
36]. DNA vaccines are simple vehicles for
in vivo transfection and antigen production leading to induction of immunity. A DNA vaccine can activate the innate immune responses by the presence of hypomethylated CpG dinucleotide sequences with particular surrounding motifs in the bacterial plasmid backbone [
38]. This may be a natural response to exposure to a bacterial DNA and is a significant operational component of DNA vaccines. However, this does not completely explain how plasmid DNA is perceived by the innate immune response. Oligonucleotides are known to require Toll-like receptor 9 (TLR-9) for immune-influencing activity, but DNA vaccines operate normally in TLR-9 -/- mice, indicating the involvement of additional receptors [
39].
In terms of induction of immunity, it is difficult to generalise about DNA vaccines. Site and procedure of injection have critical influence on the immune activation. Muscle and skin cells are clearly able to act as antigen depots. The skin contains antigen presenting cells (APCs), hence capable of priming the immune system [
40]. Roos
at al. have optimised intra-dermal EP mediated PSA DNA vaccination and effectively induced PSA-specific T cells [
41]. However, after i.m. plasmid delivery, it is likely that cross-presentation to APCs is the major route to priming [
42]. The uncertainty on this point makes rational design more difficult. A recent investigation of the route of access of exogenous phagosomes to the MHC class I pathway could have relevance. The phagosomes apparently carry elements of the endoplasmic reticulum, creating organelles capable of antigen processing for induction of cytotoxic T cell responses [
43,
44]. It is conceivable that transfected depot cells undergoing apoptosis can behave similarly. The process that conveys antigens to the APCs seems highly efficient in that DNA vaccines that produce only very low levels of antigen can induce all arms of the immune response [
45]. However, there may be different requirements for priming or boosting immunity and to activate anti tumour immunity; both processes need to be efficient. It is also essential that tumour cells alone can boost the vaccine-induced response so that continuing pressure is maintained against emergent cells. Translation of the immune therapies to clinical practice requires important optimisation. Various regimens of vaccine base therapies have been reported previously [
21,
46]. However, on review of the literature it is still not established which vaccination schedule is superior. We have shown that repeated vaccination provided optimal immunological tumour protective effects in our setting. Furthermore, repeated EP driven vaccination was safe as all immunised mice remained healthy and no adverse effect or treatment related death was observed.
The effective delivery of the vaccine vector to the host cells is a prime step for achieving immune activation. We used selected parameters of EP as a tool to boost the transfection of the muscle cells [
27]. The transfer of DNA into the cells is a process where the cells membranes are initially permeabilised and then the DNA moved by electrophoretic forces into cytosol during the following pulses. Because of this, it has been shown that small molecules can diffuse into permeabilised cells in the minutes before membrane resealing. In contrast, there is no gene transfer if the DNA is added after the pulse [
47]. Electric pulse parameters optimal for plasmid delivery (in the region of 1200 V/cm, 6-8 pulses) are known to increase gene expression 100-fold in muscle and other tissues, and have been shown to enhance humoral immune responses [
22]. The high voltage pulse was to induce electroporation in the cell membrane and the ensuing small voltage pulses were to create an electrophoretic field to assist movement of the negative charged DNA plasmid across the cells [
48,
49]. The adjuvant effects of low voltage pulses might consist of increased activation and migration of the APCs, higher transfection of relevant APCs, or increased cellular infiltration. The optimal conditions for DNA vaccination, therefore, depend on the capacity of electroporation to enhance cellular immunity, especially for cancer vaccines for which IFNγ producing CD8
+ T cells are critical. The requirements might be different for the induction of humoral immune responses, for which the induced gene expression level might be of greater importance. Muscle is the most commonly targeted tissue for vaccine delivery where gene expression may last in excess of six months. The dominant mechanism for priming of CD8
+ T cells by APCs, after DNA vaccination, is still a matter of debate and may vary depending on if DNA is delivered into the muscle or the skin.
Despite these promising effects, the clinical efficiency of the different immunotherapeutic strategies for the majority of patients with advanced prostate cancer is still limited owing to various immune evasion mechanisms mediated by tumours. One of the major challenges in developing tumour vaccines relates to the fact that as tumours grow, the immune system looses the ability to target tumour cells, because of development of several immune evading strategies. These mechanisms include down-regulation of different components of the MHC class I processing and presentation machinery, generation of antigen loss variants, production of inhibitory cytokines such as transforming growth factor-ß and IL10, and expression of apoptosis-inducing molecules [
50,
51]. DNA vaccines, such as phPSA, have potential to activate specific tumour protective immunity and have potential to overcome these tumour escape mechanisms. Hormone therapy and radiotherapy for prostate cancer can have stimulatory effects on immune system [
52,
53]. Hence, a DNA vaccine such as phPSA has potential to be used in conjunction with available treatment for prostate cancer. The translation of this vaccination in clinical trials is further supported by the work of Ottensmeier
et al. [
54]. They have shown that EP is a potent method for stimulating humoral responses induced by DNA vaccination (encoding PSMA) in prostate cancer patients. It is hoped that prostate tumour vaccines would be able to destroy tumour cells that have survived hormone-blockade or radiotherapy.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SA carried out mice experiments, performed the statistical analysis, interpreted the data, and drafted the manuscript. GC carried out the in vitro experiments and performed statistical analysis. MT and PS participated in design of the study, helped with data interpretation, and drafting the manuscript. GCOS and MT conceived the study, participated in its design, and coordinated and drafted the manuscript. All authors read and approved the final manuscript.