Some increase in the ability to diagnose and surgically treat primary brain tumors has been achieved, although the mortality and overall survival of patients with these tumors has not improved over many years [1]. The present standard treatment modalities, both surgery to remove the tumors and subsequent radiation therapy and chemotherapy, each have significant side effects. The few long-term survivors are inevitably left with cognitive deficits and other disabilities [2,3]. Gliomas are resistant to radiation and standard cytotoxic chemotherapies, making it difficult to treat these tumors [4,5]. Novel therapies are urgently needed.

Normal and malignant cells generally have antigenic differences, which are the basis for clinical immunotherapeutic strategies. Several different strategies have been attempted to enhance anti-tumor immune responses in mice and patients with intracerebral neoplasms. Immunization with dendritic cells that have been “fed” derivatives of tumor cells or transfected with tumor RNA can result in the development of immune responses against the tumor antigens expressed by malignant cells [6,7]. In patients, immunization with autologous dendritic cells, transfected with mRNA from malignant gliomas, has been found to elicit tumor-specific CD8⁺ cytotoxic T-lymphocyte (CTL) responses against the patient’s malignant cells [8]. Novel and more specific targets, such as glioma stem-like cells, have been shown to increase the success of dendritic cell immunotherapy [9]. Although the results of dendritic cell immunotherapy have demonstrated some good results in animal models, clinical trials have documented few benefits, found to be limited to a minority of treated patients [10].

Another strategy involves the preparation of a vaccine, prepared by the transfer of a cDNA expression library derived from tumor cells into an allogeneic mouse fibroblast cell line expressing a cytokine such as IL-2, which appears to have great potential for the development of an anti-tumor immune response, in the treatment of an intracerebral tumor, in mouse models [11]. Upon the transfer of the cDNA-expression library from the tumor cells into a highly immunogenic fibroblast cell line, genes specifying tumor antigens are expressed. The transferred DNA integrates spontaneously into the genome of the recipient cells and replicates as the cells divide. The transfected fibroblasts can be expanded to obtain quantities for repeated immunizations of the patient. This strategy has the capability of stimulating immunity to a broad array of tumor antigens that characterize the patient’s tumor. Only small amounts of DNA are required to prepare the vaccine, which can be obtained from tumor tissue, such that treatments can be initiated early in the development of the disease.

In many aggressive tumors, such as gliomas, progression is enhanced by local immunosuppression, associated with an increase in regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) [12]. The lack of response to treatment in glioma patients may be secondary to the immunosuppressive T cells that normally prevent an anti-tumor immune response [13]. Various cytokines, including interleukin-10 and transforming growth factor-β, have been implicated in the stimulation of Tregs. The targeting of immune checkpoints which regulate the immune system is emerging as a potent and viable cancer therapy [14]. Immunosuppressive mediators, such as IL-10, TGF-β and prostaglandin, can inhibit the function of the immune system and promote the growth of tumors [15]. Reversing the immunosuppressive tumor microenvironment is one of the keys to the success of tumor treatment.

There are several immunomodulatory cytokines, including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, which belong to the family of four α-helix bundle cytokines [16]. The development of IL-2 has been significant in the development of immunotherapy in cancer [17]. However, the use of IL-2 is limited by its toxicity risk and the expansion of regulatory T cells. The associated toxicity issues include hypertension, weight gain, hypothyroidism, cardiac arrhythmias and impaired renal function. To overcome these limitations and improve response rates, other T cell stimulatory agents, such as IL-15, have been in clinical development. IL-15 has been shown to have particularly high potential for use in immunotherapy against various tumors [18]. Furthermore, IL-15, unlike IL-2, does not contribute to the maintenance of regulatory T cells [19].

A variety of tumor vaccination strategies have been attempted, including the modification of neoplastic cells to stimulate anti-tumor immune responses. Immunization with tumor cells, modified to secrete immune-augmenting cytokines such as IL-2, IFN-γ and GM-CSF, has resulted in the development of generalized MHC-restricted anti-tumor immune responses in animal models [20,21,22,23,24,25,26,27,28]. Tumor regression has been documented in experimental animals receiving immunotherapy alone [22,23,27,28], which suggests that this treatment strategy may be effective.

Oncolytic virus is a type of virus, either engineered or in nature, which may selectively infect and lyse tumor cells while not affecting normal cells [29]. There are many oncolytic viruses, including herpes simplex virus (HSV), adenovirus, reovirus, poliovirus (PV), vaccinia virus (VV), myxoma virus (vMyx), measles virus, vesicular stomatitis virus (VSV) and Newcastle disease virus [30]. There are multiple potential mechanisms contributing to the selectivity of oncolytic viruses for tumor cells over normal cells. First, viruses can enter tumor cells by binding with certain receptors which are overexpressed on tumor cell surfaces. For instance, HSV binds with herpes virus entry mediators (HVEMs) or nectin-1, VV binds with glycosaminoglycans (GAGs) and VSV binds with low-density lipoprotein receptors (LDLRs) to enter host cells [30]. Second, some of the hyper-activated signaling pathways in tumor cells over normal cells may facilitate virus infection. Hyper-activation of AKT (serine/threonine kinase) is commonly found in most cancer cells, which is a requirement for vMyx infection [31,32]. EGFR activation, common in cancer cells, contributes to a productive infection by the attenuated vaccinia virus JX-594 [33]. Third, the deficiency of tumor cells to Type I interferon responses minimize the anti-viral immune responses, allowing oncolytic viruses to replicate [34,35]. Fourth, the dysfunction of tumor suppressor genes, such as p53, ataxia telangiectasia (ATM) and retinoblastoma protein (Rb), can potentially compromise cellular antiviral activity by accumulating genomic instability and blocking the apoptotic response [36], which contributes to the permissiveness of cancer cells.

Once oncolytic viruses infect tumor cells, they may contribute to the anti-tumor response through a direct cytotoxic effect on tumor cells and the consequent release of tumor-associated antigens, which could stimulate anti-tumor immune responses, turning a “cold” tumor into a “hot” tumor [37]. When the virus is engineered to express an immunostimulatory cytokine [38], it becomes a vector for local expression of potent immune-activating agents, attracting immune cells into the tumor microenvironment (TME) while limiting inflammation.

Many oncolytic viruses have already been tested in several preclinical and clinical trials. T-VEC (also known as Talimogene laherparepvec or OncoVEX^GM-CSF) was the first oncolytic virus approved for the treatment of advanced melanoma by the U.S. Food and Drug Administration (FDA) in 2015 [39]. T-VEC is an engineered oncolytic herpes simplex virus type 1 (HSV-1), whose neurovirulence factor ICP34.5 is replaced by the gene of human granulocyte–macrophage colony-stimulating factor (hGM-CSF) and whose viral ICP47 gene is deleted [40], to prevent neuronal involvement [41] and enhance anti-tumor efficacy. A OPTiM phase III clinical trial showed great efficacy of T-VEC in targeting patients with early metastatic melanoma (stage IIIB/C-IVM1a) [42]. It also showed enhanced anti-tumor activity when T-VEC was combined with pembrolizumab (anti-programmed death-ligand 1 antibody; PD-1 blockade) in a phase II clinical trial [43]. Furthermore, G47∆, a triple-mutated third-generation oncolytic HSV-1 [44], has a high safety profile and high anti-tumor efficacy (with a 1-year survival rate of 92.3% versus 15%) when targeting human glioblastoma in a phase II clinical trial [45].

Poliovirus is another potential candidate for oncolytic virotherapy. The recombinant nonpathogenic polio–rhinovirus chimera (PVSRIPO) is a neuro-attenuated recombinant poliovirus (Sabin vaccine strains), whose internal ribosomal entry site (IRES) was replaced with human rhinovirus type 2 (HRV2) [46]. The result from a phase I clinical trial, where 61 patients with recurrent World Health Organization (WHO) grade IV malignant gliomas were intratumorally infused with PVSRIPO, confirmed the safety of PVSRIPO when used in the brain, and the patients showed a significantly higher survival rate at 24 and 36 months after virus infusion [47]. Studies also showed that PVSRIPO has the potential to have a therapeutic effect on breast cancer, prostate cancer [48] and neuroblastoma [49].

Poxvirus, a group of large, enveloped DNA viruses associated with diseases that generate poxes in the skin, can also be a good choice for oncolytic virotherapy, since the entire replication of poxvirus happens in viral factories within the cytoplasm of infected cells, with no integration of viral DNA into the host genome, which is safe for host cells [50]. Poxviruses can take multiple large foreign genes into their genomes [51] which supports the feasibility of further arming poxviruses (e.g., adding genes of tumor antigen or immune-enhancing cytokines to poxviruses). The vvDD vaccinia virus is a new strain of poxvirus, which was attenuated by the double deletion of the thymidine kinase and the vaccinia growth factor. A preclinical study showed great anti-tumor efficacy when mice bearing MC38 colon cancer or ID8 ovarian cancer were treated with IL15-armed vvDD vaccinia virus. In addition, when combined with a PD-1 blockade, IL15-armed vvDD vaccinia virus led to dramatic tumor regression [52]. It has been reported that IL15-armed myxoma virus (another poxvirus) cured 83% of mice bearing orthotopic gliomas, when combined with adoptive T cell therapy, rapamycin and celecoxib [53].

Despite the promising results, some concerns still need attention when using oncolytic viruses. Safety issues remain a major concern for oncolytic virotherapy. For example, vvDD vaccinia virus, which has undergone two phase I clinical trials and has been found to be safe in humans [54,55], can still be fatal for hosts if it accidentally enters the cerebral lateral ventricle [56]. Therefore, it is essential to study the safety profile of the oncolytic virus in detail before moving to clinical trials. Another concern is the development of the anti-viral immune responses mediated by neutralizing antibodies [57] and immune cells, such as macrophages [58] and natural killer [59] (NK) cells.