Application of Alphaviral Vectors for

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REVIEW ARTICLE

Application of Alphaviral Vectors for Immunomodulation in Cancer Therapy Anna Zajakina1, Karina Spunde1 and Kenneth Lundstrom2,* 1

Biomedical Research and Study Centre, Riga, Latvia; 2PanTherapeutics, Rue des Remparts 4, CH1095 Lutry, Switzerland

ARTICLE HISTORY Received: April 13, 2017 Accepted: June 15, 2017 DOI: 10.2174/1381612823666170622094715

Abstract: Background: The lack of specific and efficient cancer therapies has influenced the development of novel approaches, such as immunotherapy, which from its original application of immunogenic protein delivery has developed into the use of more sophisticated recombinant gene delivery methods to achieve better safety and efficacy profiles. This approach involves viral and non-viral delivery systems. Methods: Expression vectors have been engineered for alphaviruses, including Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus. For immunotherapeutic applications, recombinant particles, RNA replicons and layered DNA vectors that express tumor-associated antigens (TAAs) and cytokines have been studied in animal models and in a few clinical trials. Results: Immunization studies with TAAs and cytokines have elicited strong antibody responses and vaccination has provided protection against challenges with tumor cells in mouse models. Furthermore, the combination of TAAs and cytokines, antibodies and growth factors and the co-administration of chemotherapeutics and bacteria-based adjuvants have enhanced immunogenicity. Intratumoral and systemic delivery of recombinant alphavirus particles has demonstrated significant tumor regression and prolonged survival rates in rodent tumor models. Conclusion: Alphavirus-based immunotherapy represents a rapid and efficient method for prophylactic and therapeutic applications in animal models.

Keywords: Alphaviruses, recombinant particles, RNA replicons, layered DNA vectors, immunotherapy, vaccines, gene therapy, cancer therapy. INTRODUCTION Cancer therapy has for a long time suffered from serious side effects and limited efficacy [1]. In attempts to address these shortcomings, one approach has been to apply viral and non-viral vectors for immunomodulation in cancer therapy [2, 3]. In this context, both intratumoral and systemic administration of vectors aimed at the delivery of anti-cancer genes, toxic genes and immunostimulatory genes have been utilized for therapeutic intervention for nonviral [2] and viral vectors [3, 4] leading to significant tumor regression and prolonged survival rates. In this review, we focus on alphavirus vectors. A summary on the biology of alphaviruses and the engineering of alphavirus expression vectors is presented. Moreover, a number of applications of alphaviruses for the immunomodulation in cancer therapy are described. Biology of Alphaviruses Alphaviruses belong to the Togaviridae family and represent a wide geographical distribution covering all continents except for Antarctica [5]. The Old World viruses including Semliki Forest virus (SFV) and Sindbis virus (SIN) have been isolated from Europe, Asia, Africa and Australia, whereas the New World viruses such as Venezuelan equine encephalitis virus (VEE) and eastern equine encephalitis virus (EEE) originate from North and South America [6]. Alphaviruses can potentially present a serious threat to the health of domestic animals and humans in many locations [7-9]. For instance, EEE and western equine encephalitis virus (WEE) have been responsible for the cause of fatal encephalitis in humans in North and South America. [10]. Typically, patients suffer from fever, anorexia, depression and clinical signs of encephalomyelitis. In both humans and horses the fatality may be as high as 90%, specifically for EEE. Lifelong immunity has been detected in *Address correspondence to this author at the PanTherapeutics, Rue des Remparts 4, CH1095 Lutry, Switzerland ; Tel: +41-79 776 6351; E-mail: [email protected] 1381-6128/17 $58.00+.00

survivors although they might develop permanent neuropathology. Furthermore, VEE can also cause human illness. Both SFV and SIN have been associated with a number of fever epidemics in Africa [11, 12]. However, the laboratory strains are considered avirulent for humans not causing any disease [13] except for one fatal case of encephalitis in a laboratory worker due to the development of enhanced virulence in laboratory animals [14]. Furthermore natural SIN variants have been found responsible for causing painful polyarthritis in Northern Europe [15]. More recently, Chikungunya virus (CHIK) has caused outbreaks in the Republic of Congo infecting thousands of people [16]. Likewise, a CHIK epidemic with painful rheumatic symptoms which saw one third of the total population of 800,000 inhabitants of the island of Reunion broke out in 2005 [17]. Unexpectedly, the East-Central-South African genotype of CHIK was discovered in 2014 in Bahia state in Brazil with over 5500 cases [18]. Alphaviruses are arthropod borne and commonly spread by mosquitoes [5]. However, SIN and EEE have also been isolated from naturally infected mites and lice [12, 19, 20]. In many cases birds are the primary vertebrate hosts for alphaviruses and for instance a South American strain of EEE was isolated from the blood of migrating birds in the Mississippi delta [21]. Alphavirus Structure and Genome Organization Alphaviruses are composed of an icosahedral nucleocapsid surrounded by a tight-fitting glycoprotein envelope [5]. The glycoproteins are fitted in an icosahedral lattice with a very regular structure confirmed by x-ray crystallization of SFV and SIN [22] and further confirmed by cryoelectron microscopy [23, 24]. The nucleocapsid contains the single-stranded RNA (ssRNA) genome and capsid protein of icosahedral symmetry. Based on x-ray diffraction and cryoelectron microscopy the nucleocapsid particles contains 240 copies and a T = 4 icosahedral symmetry (composed of 12 pentameric and 30 hexameric capsomeres) [25].

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The envelope structure of alphaviruses is composed of a lipid bilayer containing embedded viral glycoproteins [26]. Most alphaviruses have two envelope proteins, E1 and E2, anchored in the lipid bilayer through a membrane-spanning domain although a third protein (E3) is translated in the precursor PE2 (SIN) or p62 (SFV) polyprotein [4, 25]. E3 is cleaved off and only for SFV remains associated with virions with a central role for E3 in complex formation and transport of the viral envelope components to the budding site [27]. E1 and E2 form heterodimers and the interaction of three E1-E2 heterodimers results in spike structures on the viral surface [26]. An icosahedral lattice of T = 4 is formed of 240 heterodimers assembled into 80 spikes. The ratio between envelope and capsid proteins has been suggested to be one-to-one in agreement with the 240 copies of nucleocapsid protein. The small hydrophobic 6K protein is produced as a linker between E2 and E1 and has an impact on the budding process [28] and the spike structure [29]. The ssRNA genome of alphaviruses is approximately 11.7 kb containing two major regions (Fig. 1A) [4]. Two-thirds of the 5’ end region encodes for the nonstructural or replicase proteins initially translated as a polyprotein and cleaved into the nonstructural

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proteins nsP1, nsP2, nsP3 and nsP4. The 3’ end one-third of the genome is translated as a polyprotein from a subgenomic 26S mRNA, which is processed into capsid and envelope proteins. During RNA replication, initially a minus-strand copy of the full-length RNA genome is generated, which serves as a template for the production of full-length genomic RNA and also transcription of subgenomic RNA [30]. Life-cycle of Alphaviruses Although alphaviruses can replicate in both arthropod and vertebrate hosts there is a huge difference as persistent lifelong infection prevails in arthropods while the infection in vertebrates is characterized by an acute short-time infection [5] (Fig. 1B). Similarly, alphaviruses generate a chronic infection in mosquito cells while cytotoxicity and inhibition of host cell protein synthesis in vertebrate cells results in apoptosis and rapid cell death. Alphaviruses possess a broad host range in both invertebrates (mosquitoes or other hematophagous insects) and vertebrates (mammals, birds, amphibians, reptiles) [7, 32]. The virus replication occurs in a wide variety of cells including neurons and glial

Fig. (1). Genome structure and life-cycle of alphaviruses. A. Alphavirus genome structure. The ssRNA genome comprises the nonstructural and structural genes. B. Production of viral progeny from RNA genome. Once RNA is released into the cytoplasm, a negative strand RNA is synthesized as a template for RNA replication and translation of nonstructural proteins (replicase complex) and structural genes from the subgenomic 26S promoter takes place. RNA genomes are packaged into nucleocapsids and the envelope proteins are transported to the plasma membrane, where assembly and budding of mature viral particles takes place. Republished with permission [31].

Application of Alphaviral Vectors for Immunomodulation in Cancer Therapy

cells, striate and smooth muscle cells, lymphoid cells, synovial cells and brown fat cells [7]. One of the key questions is which host cell receptors are targeted by alphaviruses. One early indication of the nature of SIN receptors was the reduction of virus binding after protease treatment, but not neuraminidase or phospholipase treatment [33]. Furthermore, it was demonstrated that a neurovirulent and an avirulent SIN strain did not compete with one another for binding and therefore use completely different receptors. Additionally, two different neuronal cell lines possessed many more receptors for the neurovirulent strain than for the avirulent strain suggesting that the nature of the receptors appeared to affect virulence. Antigens of the MHC, HLA-A and HLA-B in humans and H-2K and H-2D in mice have been suggested as receptors for SFV as these antigens bind to SFV glycoproteins [34]. Furthermore, an anti-idiotypic SIN E2 antibody 49 showed binding to chicken cells, blocked 50% of virus binding and immunoprecipitated a 63 kD protein from chicken plasma membrane preparations indicative of a putative SIN receptor [35]. Generation of anti-receptor monoclonal antibodies, which block SIN binding to BHK cells, revealed that mAb 1C3 binds to the C-terminus of the high affinity laminin receptor [36]. Furthermore, two laboratory strains of SFV and SIN have been shown to target heparan sulfate receptors [37]. In contrast, Ross River virus (RRV) does not significantly utilize heparan sulfate as a receptor, but a single amino acid substitution at residue 218 in the RRV E2 glycoprotein resulted in heparan sulfate binding and expansion of the host range to chicken embryo fibroblasts [38]. Electron cryomicroscopy of the RRV E2 N218R mutant and heparin complex demonstrated heparin binding to the distal end of RRV spikes. In another study, the importance of the interaction of the E2 protein with heparan sulfate was confirmed for SIN [39]. As the SIN-like XJ-160 virus failed to infect mouse embryonic fibroblasts (MEFs), the XJ-160 E1 and E2 genes were replaced by the corresponding SIN sequences, which resulted in chimeric viruses available of infecting MEFs. Further studies in MEFs producing shortened heparan sulfate chains demonstrated an essential role for the E2 glycoprotein in cellular infection. After successful binding to a receptor, the alphavirus envelope fuses with the cellular membrane and the nucleocapsid is released to the cytoplasm. A highly conserved hydrophobic domain of the E1 glycoprotein has been associated with fusion activity [40]. Alphaviruses enter host cells through endocytosis in clathrin-coated vesicles followed by transfer to endosomes due to low pH leading to conformational reorganization of E1-E2 heterodimers [41]. Furthermore, it was demonstrated that the sodium concentration in the medium was important for virus entry suggesting that fusion also depended on the maintenance of a voltage potential [42]. In contrast to the model supporting endocytosis, it has been suggested that alphaviruses can infect cells by direct fusion to the cell surface [4345]. Nucleocapsids are dissembled in the cytoplasm as capsid protein is associated with ribosomes releasing the RNA genome [46]. After the initial replication of a minus-strand copy of the RNA genome full length and 26S RNA are generated to provide nonstructural and structural proteins for new viral progeny [5]. The polyprotein nsP1234 is cleaved into the individual nsp-4 proteins forming the RNA replicase complex and the structural proteins are translated from the last third (26S RNA) of the RNA genome [30, 32]. Viral “replication factories” are formed at cytopathic vacuoles (CPVs) as sites for RNA replication and translation and nucleocapsid assembly [47]. Immuno-electron microscopy has suggested that CPVs are derived from endosomes and lysosomes and the nonstructural proteins (replicase complex) are associated with the cytoplasmic surface of the CPVs [48]. The host cell protein synthesis is abruptly inhibited approximately 3 h after infection due to competition for the translational machinery by viral mRNA, alteration of the environment favoring viral mRNA translation, direct inhibition by the viral capsid protein

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and interference with initiation factors caused by the nonstructural proteins [49]. However, the subject of inhibition of host protein synthesis is complex and early viral gene function yet to be determined might be involved. Once RNA replication has taken place and capsid protein has been synthesized, assembly of nucleocapsids occurs [5]. For SIN a packaging signal has been detected in the nsP1 gene (nt 746-1226) [50], whereas the packaging signal for SFV is located in the nsP2 gene [51]. In contrast, the packaging signal for AURA virus, serologically related to SIN, is located in the 26S region [52]. Although packaging signals are located in different positions for SIN, SFV and AURA the signals appear to be related to one another. When the RRV capsid protein was replaced by the SIN capsid the RNA of the chimeric virus was efficiently encapsidated [53]. It seems that related to the assembly of nucleocapsids genomic RNA is encapsidated and no RNA-free viral particles are generated. The translated alphaviral glycoproteins are initially folded immediately upon entry into the endoplasmic reticulum (ER) requiring molecular chaperons, folding enzymes and the formation of disulfide bonds [54]. Incorrectly folded proteins are not transported out of the ER and usually aggregate into non-specific complexes followed by degradation. Both PE2-E1 and E2-E1 heterodimers are formed, which are transported to the Golgi complex [55]. Glycoproteins are then transported from the trans Golgi network to the plasma membrane, where the assembly of viral particles occurs. SFV or SIN infection of thyroid epithelial cells has shown accumulation of viral glycoproteins at the apical surface, where also the budding occurs [56, 57]. In contrast, in SFV-infected Madin-Darby canine kidney (MDCK) cells [58] and SIN-infected Caco-2 cells [57] the glycoproteins are found on the basolateral surface, which is also the site of budding. Furthermore, the viral glycoproteins were exclusively present in dendrites and cell bodies but not in axons in SFV-infected hippocampal cells [59]. During the transport, the PE2 is cleaved into E2 and E3 by host cell proteinases between the Golgi network and the plasma membrane in vertebrate cells [60], whereas it occurs early in mosquito cells [61]. Before the release of viral particles occurs, the C-terminal of E2 is re-orientated into the cytoplasm [62]. For the budding process of virions, nucleocapsids assembled in the cytoplasm diffuse freely to the plasma membrane, where interaction with the glycoproteins occurs. It has been postulated that the nucleocapsid binds specifically to the C-terminus of the cytoplasmic domain of the E2 glycoprotein [62] and that the binding provides free energy, which translocates the capsid through the plasma membrane. Alphavirus Expression Systems Generally, the expression vectors which have been engineered are based on attenuated or avirulent alphavirus strains to guarantee the highest possible biosafety levels. Alphavirus systems have been in use for the last 25 years for the expression of a large range of recombinant proteins in mammalian cells lines [63], primary cells [64] and in vivo [65]. The most commonly used alphavirus expression vectors are based on SFV [9, 63], SIN [66] and VEE [67]. A number of topologically different proteins have been expressed from SFV vectors [68]. Particularly, expression of integral membrane proteins in various mammalian cell lines has allowed functional studies in support of drug discovery [69]. In relation to vaccine development, vectors based on SFV, SIN and VEE have been applied as RNA replicons, recombinant particles and layered DNA vectors for immunization of animals against viral and tumor antigens [70]. Vaccinations have resulted in generation of neutralizing antibodies and in several cases protection against challenges with tumor cells or lethal doses of viruses. Generally, vectors have been engineered for the delivery of recombinant genes in three different formats as recombinant viral particles, RNA replicons and layered DNA plasmid vectors (Fig. 2).


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Fig. (2). Alphavirus-based expression systems. 1. RNA delivery. In vitro transcribed RNA from the alphavirus expression vector is electroporated or transfected into mammalian host cells, where cytoplasmic RNA replication results in high-level expression of the gene of interest. 2. Recombinant particle delivery. Host cells are infected with recombinant alphavirus particles leading to the release of RNA, RNA replication and high-level expression of the gene of interest. 3. DNA delivery. The alphavirus DNA expression vector is transfected into host cells, delivered to the nucleus and transcribed RNA translocated to the cytoplasm for RNA replication and high level of expression of the gene of interest. Republished with permission [31].

Very similar expression systems have been developed for SFV, SIN and VEE, which also falls into the following three categories. Replication-deficient vectors are composed of an expression vector containing the alphaviral nonstructural genes nsP1-4, the subgenomic 26S promoter and the gene of interest (Fig. 3). The nsP1-4 proteins produce replicase complexes responsible for high-level RNA replication and when such replicon RNA is introduced into cells, generation of high quantities of recombinant protein is achieved. When alphaviral structural proteins are provided in trans from a helper vector, high-titer replication-deficient particles can be generated from in vitro transcribed RNA from expression and helper vectors co-transfected into mammalian host cells. Replication-proficient vectors are based on the full-length alphavirus genome in which a second subgenomic promoter has been inserted either upstream or downstream of the alphavirus structural genes (Fig. 3). In vitro transcribed RNA from these vectors generates recombinant protein expression and simultaneous production of replication-proficient recombinant particles. Layered DNA vectors are engineered by replacing the SP6 RNA polymerase promoter with a CMV promoter and are directly used as plasmid DNA for tranfection of host cells. This approach is highly dependent on available transfection methods, but eliminates the risk of generation of any virus progeny. However, co-transfection of layered DNA expression vectors and DNA helper vector produces recombinant particles, although at 10-100 fold lower titers compared to application of in vitro transcribed RNA [71]. Moreover, transfection of engi-

neered full-length layered DNA vectors generates replicationproficient alphaviral particles. The choice of alphavirus vector system is to some extent dictated by the chosen application. In case of large quantities of highlevel expression of recombinant protein, the obvious choice is replication-deficient particles. Furthermore, expression in primary neurons [64] and local in vitro delivery [65] favors the use of replication-deficient particles. In case of extended spread and duration of expression, replication-proficient vectors should be considered [72]. Immunization studies have been carried out with RNA replicons, replication-deficient particles and layered DNA vectors [70], which all have shown efficacy as described below. To facilitate production of recombinant alphavirus particles, especially at large scale, packaging cell lines have been established. For instance, a panel of alphavirus vector packaging cell lines has been engineered, in which expression cassettes constitutively producing the SIN structural proteins were stably transformed [73]. Translation of the structural proteins was inducible and occurred only when cells were transfected with an alphaviral vector encoding the replicase genes. One construct separated the capsid and envelope glycoproteins onto different expression cassettes, which eliminated the generation of recombinant-competent virus, but resulted in modest titers of 107 infectious units/ml. The packaging cell line system allowed both SIN and SFV particle generation with similar



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Fig. (3). Alphavirus-based recombinant particle production. In vitro transcribed RNAs from alphavirus expression and helper vectors are electroporated or transfected into mammalian host cells (generally BHK-21 cells). After RNA replication and expression of alphavirus proteins, recombinant RNA is packaged into nucleocapsids due to the presence of a packaging signal (PS) only on the recombinant RNA. After transport of envelope proteins to the plasma membrane assembly and budding of recombinant particles takes place. Republished with permission [31].

efficiency. In another approach, a packaging cell line was engineered for the SIN-like XJ-160 virus [74]. Two cassettes with the capsid and envelope glycoprotein genes were stably transfected into BHK-21 cells and selection with G418 and hygromycin carried out. The packaging cell line allowed production of high titer XJ-160 and SFV recombinant particles at large scale. 2.1. Expression Vector Development In attempts to enhance the expression levels, reduce the cytopathogenic effects and prolong the duration of expression a number of modified expression vectors have been engineered [75] (Fig. 4). Several point mutations have been introduced into the nsP2 and nsP4 (nonstructural) genes of both SFV [75, 76] and SIN [77, 78] vectors. Generally point mutations in the nsP2 gene provide reduced cytotoxicity of host cells and thereby prolonged transgene expression [75]. Moreover, the SFV-PD vector containing two point mutations in the nsP2 gene showed enhanced expression levels in a broad range of mammalian host cells [76]. In another study, a single point mutation in the SIN nsP2 gene generated persistent infection of transduced host cells [79]. Furthermore, expression vectors have been engineered from avirulent alphavirus strains. For instance, introduction of the replicase genes from the avirulent strain SFV A7(74) showed significantly reduced cytotoxicity in comparison to the conventional SFV vector in mammalian cell lines and primary neurons [80]. Moreover, the expression profile was clearly temperature-dependent and its duration was extended. In another approach, point mutations were introduced into the SIN capsid gene (Ser 180/Gly 183), which resulted in production of exceptionally large viral particles of 205 nm in comparison to the normal size of 70 nm [81]. It was possible to package up to 18 kb RNA without any significant titer reduction 109 pfu/ml) with the potential capacity of 32 kb. Another approach to improve expression levels has been to introduce the translation enhancement signal

(TES) of the capsid protein into the SFV expression vector, which can enhance transgene expression levels 5-10 fold [82]. Furthermore, when the FMDV 2A protease sequence was introduced downstream of the capsid TES proper cleavage of the capsid sequence from the final product occurred [83]. Enhanced expression levels of transgene expression have also been achieved by the design of VEE self-replicating subgenomic RNA replicons [84]. This approach allowed amplification of underused VEE replicon enzymes and provided 10-50 fold improvement in protein expression levels. Alphavirus helper vectors have also been subjected to engineering exercises in attempts to increase the biosafety of applications of alphavirus recombinant particles (Fig. 4). The main point to address is to limit or preferentially exclude recombination events between expression and helper vector RNA, which will generate replicationproficient particles. Helper vector engineering is described in detail in the section on biosafety. Comparisons of Alphaviruses to other Viral Vectors In addition to alphaviruses, obviously other virus-based vectors such as adenoviruses, AAV, flaviviruses, herpes simplex viruses, lentiviruses, measles virus, retroviruses and rhabdoviruses have been applied for cancer therapy and vaccine development [85]. However, it is not the topic of this review to compare different viral vectors to each other, so only a short description of each system is presented. Adenovirus vectors are the most commonly used viral vectors, which have been subjected to a number of preclinical and clinical trials [86, 87]. Much attention has been paid to vector development to develop second and third generation vectors with improved biosafety profiles and reduced pre-existing immunity in humans [88]. Evidently, appropriate methods including packaging cell lines have been developed for adenoviruses. Several studies in animal models


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Fig. (4). Examples of alphavirus expression systems and vector development. Among RNA-based vectors replication-deficient vectors include conventional cytotoxic and less cytotoxic mutant versions. Different types of helper vectors exist including pSFV-Helper2 vector, which generates conditionally infectious particles and split helper vectors, which eliminate recombination events between expression and helper vectors. Replication-proficient vectors are based on the avirulent VA7(74) strain. DNA vectors include the SIN less cytotoxic and temperature-sensitive mutant versions. Republished with permission [31].

have demonstrated tumor regression in animal models for ovarian [89], prostate [90] and brain [91] cancers. Adeno-associated virus (AAV) vectors have been frequently used due to the lack of pathogenicity and toxicity and their ability to transduce both dividing and non-dividing cells [92]. Moreover, AAV provides long-term transgene expression and has demonstrated good safety profiles in clinical trials. However, a short-

coming is the immune responses elicited after re-administration of AAV, which can be avoided by application of different AAV serotypes for repeated injections [93, 94]. Related to cancer therapy, AAV used for intravitreal administration of vectors expressing interferon-β (IFN-β) provided potent anti-tumor responses in treatment of retinoblastoma [95]. AAV serotype 2 vectors have been applied for the expression of vascular endothelial growth factor (VEGF), which prevented growth of pulmonary metastases of 4T1


tumors after intravenous administration in a mouse model [96]. AAV vectors have furthermore been subjected to oral and intramuscular vaccinations, which extended survival and provided long lasting tumor protection in 80% of immunized mice [97]. Flaviviruses, such as Kunjin virus have been applied for the expression of the granulocyte colony-stimulating factor (GM-CSF) in mice with established CT26 colon carcinoma and B16-OVA melanoma [98]. Intratumoral administration cured approximately 50% of immunized mice. In another study, Kunjin virus-based expression of the CTL epitope of human papilloma virus (HPV) E7 protein induced E7-directed T cell responses and showed protection when mice were challenged with E7-expressing epithelial tumors [99]. Herpes simplex virus (HSV) presents an attractive alternative for gene therapy applications due to the large packaging capacity of foreign DNA, attenuated oncolytic activity and establishment of life-long latent infection in neurons [100]. Related to cancer therapy, the lytic HSV-1 RH2 vector was evaluated in a syngenic C3H squamous cell carcinoma (SCC) mouse model, where tumor regression was observed even in contra-lateral tumors [101]. Moreover, oncolytic HSV-1 carrying four copies of miRNA-145 selectively reduced cell proliferation and prevented colony formation of nonsmall cell lung cancer (NSCLC) cells [102]. Furthermore, oncolytic HSV expressing the human sodium iodide symporter (NIS) enhanced anti-tumor efficacy [103]. Retroviruses have also been applied in gene therapy, but have seen some restrictions due to random integration of therapeutic genes into oncogene regions causing leukemia in retrovirus-treated patients [104, 105]. This problem has been addressed by engineering recombinant bi-functional retrovirus vectors displaying an scFV antibody to the carcinoembryonic antigen (CEA) and expressing the iNOS gene, which resulted in a significant inhibition of MKN-45 tumor growth [106]. Moreover, retrovirus vectors expressing the TNF-related apoptosis inducing ligand (TRAIL) gene showed efficient transduction of drug-resistant A2780/DDP ovarian carcinoma cells in vitro [107]. Furthermore, when the retrovirus transduced cells were subjected to cisplatin, improved anti-tumor activity was observed. Lentiviruses, belonging to retroviruses, have also been applied for cancer therapy [108, 109] fairly recently by targeting dendritic cells (DCs) for the development of a novel tumor vaccine prostate cancer model [110]. In this context, lentivirus vectors preferentially delivered the prostate stem cell antigen (PSCA) antigen to DC_SIGN expressing 293T cells and bone marrow-derived DCs. Protection against lethal challenges was obtained in a prophylactic prostate cancer model and tumor growth was reduced after treatment with a lentivirus expressing the PSCA [110]. Recombinant lentivirus vectors expressing the Wtp53-pPRIME-miR30-shRNA have also been delivered to AFP-positive liver cancer cells showing inhibition of proliferation of Hep3B cells both in vitro and in vivo [111]. Lentivirus vectors have also been applied for the delivery of shRNA of the apoptosis inhibitor protein Livin, which resulted in induced apoptosis in mice with lung adenocarcinoma tumor xenografts [112]. An interesting approach of oral lentivirus delivery of a triple mutant of osteopontin (OPN) as aerosols demonstrated inhibition of lung tumorigenesis in mice [113]. Measles virus vectors have been subjected to immunization studies, particularly the Edmonston-B (MV-Edm) strain for a number of cancer indications [114-118]. In this context, intratumoral injection of MV-Edm into SCID mice with implanted lymphoma xenografts resulted in significant tumor regression [115]. Moreover, epidermal growth factor receptor (EGFR)-retargeted MV strains efficiently targeted EGFR-expressing glioblastoma multiforme [116]. In another study, intratumoral administration of MV vectors expressing CEA demonstrated tumor growth delay and improved survival in a subcutaneous PC-3 xenograft model [114]. Also, stud-


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ies on the delivery of MV-CEA into mice with implanted MDAMB-231 mammary tumors revealed a significant delay in tumor growth and prolonged survival of immunized animals [117]. Coadministration of MV-CEA and MV-NIS (thyroidial sodium iodide symporter) in mice with ovarian tumors showed better tumor regression than with either MV construct alone [118]. Rhabdoviruses, especially Vesicular stomatitis virus (VSV) vectors with no pre-existing immunity in humans, have also been evaluated for cancer therapy [119]. For instance, oncolytic VSV vectors have been tested in human pancreatic cancer cell lines revealing superior responses to Sendai virus and RSV vectors [120]. In another study, VSV vectors expressing ΔM51 or wild type matrix protein were evaluated for apoptosis activation in aggressive pancreatic ductal adenocarcinoma (PDAC) [121]. In cells with defective IFN signaling robust apoptosis responses were observed. However, cell lines constitutively expressing high levels of IFNstimulated genes (ISGs) showed resistance to apoptosis. In comparison to other viral vectors, alphaviruses possess several favorable features. High titer virus stocks can rapidly be generated, although large-scale production still struggles due to the lack of efficient packaging cell lines. Alphavirus vectors also allow delivery of RNA replicons, recombinant replication-deficient and competent particles as well as DNA plasmids, which enhances the application possibilities. The packaging capacity of foreign genes is relatively good (up to 8 kb inserts). The expression levels of recombinant proteins are extremely high from alphavirus vectiors and when delivered as RNA replicons or recombinant particles there is no risk of genomic integration. The transient nature of expression (5-7 days in vivo) can be considered as an advantage in the context of cancer therapy and vaccinations, but obviously in cases where long-term expression is required it is a drawback. However, in contrast to for instance AAV, the low immunogenicity of alphaviruses vectors has allowed for repeated administration. One issue of worry has been the relatively high mutation rate common for replicating viral RNA [5]. However, expression of a large number of recombinant proteins from alphavirus vectors has proven highly successful and has not supported the raised concerns [85]. 3. IMMUNOTHERAPEUTIC APPROACHES One of the primary functions of the immune system is to identify and eradicate altered cells. Immune control and cancer eradication includes the activation of non-specific innate immune responses, such as natural killer (NK) cells, macrophage polarization, and specific immune responses based on antigen presentation by DCs with subsequent activation of T cells. [122]. The immune system possesses all the components required for the specific recognition of individual malignant cells, is broadly represented in the body and employs multiple mechanisms to identify and destroy tumors. However, attempts to harness the immune system’s capacity to treat cancer have had limited success. Elucidating the mechanisms by which tumors escape immune control and developing agents that target those mechanisms has resulted in novel efficient immunotherapies and renewed enthusiasm for the use of immunotherapeutic approaches to treat cancer. These approaches include cancer vaccines, oncolytic viruses, immune checkpoint antagonists, stimulatory cytokines, and different types of cellular therapies. The use of viral gene therapy vectors for cancer immunotherapy has the potential to provide specific, non-toxic options that increase tumor antigenicity and the immunogenicity of antigen-specific vaccines and modulate the tumor microenvironment by locally delivering cytokines to restore anti-cancer immune function. In addition to the natural oncolytic properties of some viruses, recent virus-based technologies have significantly contribute to the development of targeted therapeutic gene delivery approaches using viral vectors. Interest in recombinant viruses as immunostimulatory agents increased after the first viral drug for melanoma treatment based on an oncolytic herpes simplex virus (HSV) encoding granulocyte-


macrophage colony-stimulating factor (GM-CSF) was approved by the US Food and Drug Administration Agency in 2015 [123]. However, novel strategies that address the limitations of virus-based therapy, such as anti-vector pre-immunity, gene toxicity, the inefficiency of targeted delivery and insufficient levels of transgene expression, are required to improve inefficient therapeutic outcomes. The immune system’s capacity for recognizing altered antigens of malignant cells is crucial for immune therapy of cancer. Paradoxically, diminished or absent antigenicity can evolve in cancer cells due to immune selection of mutated cells that are deficient in immunogenic tumor antigens. The mechanisms by which tumor cells evade detection by the adaptive immune system include defects in expressing major histocompatibility complex (MHC) class I or low-molecular-weight protein (LMP) family molecules in malignant cells [124]. Therefore, the main goal of antigen-specific cancer therapies, such as therapeutic anti-cancer vaccines, is to induce immune cells to target cancer cells that express “non-self” antigens. Cancer vaccines depend on the efficiency of antigen uptake and DC presentation to tumor-specific CD8+ and CD4+ T cells. Thus, the choice of target antigen is of critical importance. Several phase III trials of therapeutic cancer vaccines are in progress, including some based on viral vectors, e.g., TG4010, a promising poxvirus vectorbased vaccine encoding the tumor-associated antigen Mucin-1 and IL-2 that is being tested for the treatment of non-small-cell lung cancer [125]. Due to tumor-derived immunosuppressive/anti-inflammatory signals and frequent deficiencies in the immune system for inducing the productive inflammation required to promote effective immune responses against malignant cells, it is important to enhance immunity in general or decrease the immunosuppressive action of tumor cells and their surrounding microenvironment. Non-antigen-specific therapies include cytokines, immune growth factors and immunologic adjuvants. Several strategies have been used to break immune tolerance and systemically administer cytokines to enhance the immune response (e.g., IL-2, IL-12, IFN-γ) [126]. Because of the significant side effects associated with prolonged cytokine use, cytokine administration directly into tumors or strategies to restore cytokine imbalance in a localized manner with virus vector use may be less toxic and more productive. The intrinsic property of alphavirus replication is the formation of double stranded (ds) RNA intermediates, which induce a strong innate antiviral response [127]. DsRNA can also directly activate the Toll-like receptor (TLR) 3 of DCs, enabling the induction of both innate and adaptive immune responses [128]. Moreover, alphavirus RNA replication and the appearance of dsRNA intermediates induce a shutdown of cellular protein synthesis, antiviral response and, ultimately, cell death [5]. In turn, lysis of infected tumor cells results in the release of tumorassociated antigens that further enhance anti-tumor immune responses. Although cancer cells are genetically unstable and employ multiple mechanisms to evade the immune response, changes in the cancer microenvironment may be common in many types of tumors, suggesting the possibility of therapeutic targeting of the tumor-supporting environment to treat different types of cancer. One widely explored therapeutic strategy to disrupt the tumorsupporting microenvironment is to block the dissemination of cancer cells by inhibiting the development of blood and lymphatic circulatory systems in tumors [129]. The expression of angiogenesis-inhibiting factors, such as angiostatin and endostatin, can block metastatic spread and growth. A recent study also demonstrated that expressing endothelial-derived thrombospondin-1 inhibited the metastatic dissemination of breast tumor cells, whereas metastatic growth was associated with endothelial cells producing periostin and TGF-β [130]. Several antiangiogenic treatment strategies using alphaviral vectors have been evaluated (see section 3.4 below: VEGFR-2 + cytokines).

Zajakina et al.

The down-regulation of chemokines that induce tumor infiltration by immune effector cells and the up-regulation of chemokines that attract suppressor cells, such as regulatory T cells, suppressive plasmacytoid DCs, and myeloid-derived suppressor cells, in cancer cells must be considered in therapeutic vaccine design [131]. Thus, a therapeutic vaccine must either induce anti-cancer T cells despite spontaneously expressing tumor-supporting chemokines or be administered as part of a combination therapy to alter the chemokine profile in the tumor microenvironment. An alternative anti-cytokine therapy for modulating the tumor microenvironment is the expression of cytokine receptor antagonists, such as a mutated form of a cytokine that can bind to a specific receptor but cannot induce a signal. Small inhibitory peptides may also be used for such purposes. For example, a member of the small leucine-rich repeat protein family NPN, which possesses TGF-β suppressor activity, might be expressed as a potential TGF-β inhibitor [132]. Although regulation of the tumor microenvironment has been studied extensively in cancer research for the past five years, studies have only recently examined alphaviral and other viral vectors for targeted modification of the supporting phenotype of tumorassociated immune cells (see section Combinations of anti-cancer therapeutic strategies). The composition of the tumor microenvironment has prognostic value for clinical outcomes in a variety of cancers [133]. Infiltration of tumors by CD8+ T cells is correlated with improved survival in colorectal, breast and other solid cancers [134-136]. By contrast, infiltration of macrophages and T regulatory cells (Tregs) is generally associated with inferior outcomes [137, 138]. The composition and spatial organization of tumor stroma cells also have a major impact on the response to cancer immunotherapy agents. Tumors may express cytokines (e.g., increased VEGF, TGFβ, IL-10, IL-6, and COX-2 and reduced IL-4, IL-12, IFN-α, IFN-γ, and GM-CSF), soluble factors such as transforming growth factor-β (TGF-β), VEGF or inhibitory ligands, such as PD-L1, and other molecules, such as indoleamine 2 and 3dioxygenase, that alter the polarization of natıve T cells into Tregs and actively suppress DC maturation, thus providing growth and survival signals for promoting tumorigenesis [139, 140]. Furthermore, tumors may also produce chemokines, such as CCL17 and CCL22 that induce infiltration by immunosuppressive Tregs via CCR4 signaling, as demonstrated for cutaneous lymphoma and ovarian cancer [141, 142]. Interestingly, many tumors and infiltrating macrophages appear to produce galectins that belong to the family of “galactose binding proteins”, which is hypothesized to promote the sequestration of T cell receptors (TCRs) and co-stimulatory CD8+ molecules. The subsequent dissociation of TCRs and CD8 molecules might be responsible for the resulting T cell anergy in tumors [143]. Therefore, combining vaccination with treatments that neutralize the effect of galectin may be another strategy for enhancing the antitumor immune response. Macrophages may exhibit pro- or antitumor effects depending on whether they express M2 or M1 phenotypes [144]. Tumorassociated macrophages generally express the M2 phenotype, which is likely due to polarization by IL-4, IL-10, IL-13, and/or GM-CSF in the tumor microenvironment. In contrast to the M1 phenotype, M2 macrophages promote the formation of denser stroma and lead to poor T cell infiltration. It is important to account for the key molecular differences between cells under normal and altered tissue homeostasis because there is a delicate balance between their tumor-inhibitory and tumor-promoting functions. Among recombinant viruses, alphaviral vectors are good candidates for cancer gene therapy due to their ability to mediate strong cytotoxic effects by inducing p53-independent apoptosis, efficiently overcoming immunological tolerance by activating innate antiviral pathways and subsequently triggering cytotoxic T-lymphocyte


(CTL) responses against tumors [145-147]. The advantages of alphaviral vectors also include a low specific immune response against the vector itself, absence of gene toxicity, and lack of vector pre-immunity in the majority of humans [83]. The oncolytic properties of alphaviruses can be enhanced by inducing adaptive anticancer immunity, such as by expressing tumor-associated antigens (TAA) and immune modulating chemokines. The combination of alphaviral vectors with advanced chemotherapeutics and checkpoint inhibitors has recently become a promising strategy for cancer treatment. 3.1. Oncolytic and Cytopathic Properties of Alphaviruses in Animal Models Alphaviral vectors possess oncolytic properties that destroy tumors via oncolysis. Viral replication and induction of apoptosis in cancer cells can lead to the release of TAAs after uptake by antigen-presenting cells (APCs) and the stimulation of immune responses against tumors. Earlier studies demonstrated that apoptosis is induced by SFV vectors in a p53-independent manner [148]. Because many tumors have lost p53 function, alphavirus vectors are therefore promising for overcoming the anti-apoptotic state related to p53-deficiency. Furthermore, a replication-deficient SFV vector that expresses EGFP (enhanced green fluorescent protein) inhibited tumor growth and, in some cases, led to complete tumor regression in human non-small cell lung carcinoma xenograft models [149]. This effect was mediated by p53-independent apoptosis and necrosis and required repeated intratumoral administration (three to six injections) and high doses of the vector (1 × 1010 IU/ml). Moreover, SIN vectors may be capable of targeting tumors upon systemic injection [150-153]. Similar to the SFV vector, an oncolytic effect was observed for replication-deficient SIN vectors expressing reporter genes such as LacZ or luciferase. Moreover, SIN vectors systemically and specifically targeted metastasized tumors in the peritoneal cavity and significantly suppressed tumor growth in ovarian tumor-bearing xenograft models [151]. Recently, mediastinal lymph nodes (MLNs) were identified as a site of early transient heterologous protein expression after intraperitoneal injection of SIN vectors, which generate effector and memory CD8+ T cells against TAAs (see the section on Expression of tumor-associated antigens). The antitumor effect can be increased by expressing proapoptotic genes in apoptosis-resistant tumors. In an early study, immunodeficient mouse models with established rat prostate tumors overexpressing the Bcl-2 oncogene were treated with intratumoral injections of SFV VLPs encoding the pro-apoptotic gene Bax, which plays a key role in programmed cell death [154]. Expression of the Bax gene by the SFV1 vector enhanced its cytopathic effect and led to a significant 47% reduction in tumor volume compared to untreated controls. However, complete tumor regression was not achieved in this study. In immunocompetent models, an immune response is involved in the elimination of tumors by oncolytic self-replicating vectors. The role of the immune system in response to alphaviral vectors was supported by the increased potency of the antitumor effects when the animals were pre-immunized with the same vectors. The virulent SFV4 strain and its derivative recombinant vector (SFVp62-6k), which contains deletions of the viral structural capsid and E1 genes, were used to stimulate an immune response in a CT26 colon cancer model [155]. Direct intratumoral injections of virulent SFV4 or replication-deficient VLPs induced intense inflammatory reactions and had a significant effect on animal survival. Interestingly, no differences in inhibition of tumor growth were observed between VLP- and SFV4-treated animals. Although replication-deficient alphaviral vectors expressing reporter genes have been shown to significantly inhibit tumor growth in animal models, replication-competent vectors may have additional advantages, such as prolonged transgene expression,


9

efficient vector dissemination within the tumor and enhanced tumor oncolysis due to progeny virion synthesis. One problem with the therapeutic use of oncolytic viruses is biosafety. Although alphaviral vectors that are based on SFV and SIN have low pathogenicity in humans and cause only mild disease [5], most oncolytic studies using alphaviral replication-competent vectors have been performed with avirulent SFV and SIN strains, which have a superior safety profile for clinical applications (Table 1). The avirulent SFV strain A7 expressing EGFP (SFV-VA7EGFP) has been applied intratumorally, intravenously and intraperitoneally as a therapeutic vaccine for human melanoma-bearing SCID xenografts [156]. SFV-VA7 vector inoculation resulted in significant tumor regression, regardless of the route of administration. The neurotropism of SFV did not restrict its ability to target melanoma tumors because SFV-VA7 reduced tumors to a level far below that of the initial volume within 3 weeks. Although inhibition of tumor growth dynamics was observed, small groups of actively dividing tumor cells were detected within strands of connective tissue, indicating the potential for tumor remission in the future. In another study, oncolytic virotherapy with the SFV-VA7-EGFP vector also indicated a good safety profile and resulted in almost complete inhibition of tumor growth in subcutaneous human lung adenocarcinoma in NMRI nu/nu mouse models after intratumoral vector injections [157]. Remarkably, systemic administration resulted in only delayed tumor growth (intravenous injection) or total absence of a response (intraperitoneal injection). The same study investigated the antitumor capacity of the SFV-VA7 vector in an immunocompetent rat glioma tumor model (intracranial BT4C glioma in BDIX rats). Although SFV-VA7 is a neurotropic virus, neither intravenous nor intraperitoneal systemic administration provided significant therapeutic efficacy in glioma-bearing rats. By contrast, direct intratumoral injections of SFV-VA7 led to a significant reduction in tumor growth. However, these positive results were followed by an accelerated increase in tumor growth eventually resulting in animal death [157]. The insufficient efficacy of the systemic treatment of subcutaneous lung carcinoma was partially attributed to the less optimal microenvironment of subcutaneous tumors. A subsequent study assessed the influence of the physiological tumor microenvironment on SFV-VA7-EGFP-based therapy in orthotopic human lung A549 tumors in immunodeficient NMRI-foxn1 nu/nu mice [158]. SFVVA7-EGFP-based therapy was compared to oncolytic adenovirus Ad5-D24TK-GFP administration, and tumor growth and responses to virotherapy were monitored by small-animal computerized tomography. Both oncolytic SFV-VA7-EGFP and Ad5-D24TK-GFP induced tumor cell destruction when administered in anatomical proximity to the tumors. This treatment significantly prolonged the survival of immunized animals compared to controls. However, despite the orthotopic tumor microenvironment of the lung cancer xenografts, oncolytic efficacy was limited when the vectors were systemically injected. This inefficiency of achieving a cure in vivo may be associated with strong IFN-α/β responses to virus infections in A549 tumors. Interestingly, although the SFV-VA7 vector demonstrated insufficient efficacy in the systemic treatment of brain tumors in immunocompetent glioma-bearing rats [157], systemic inoculation of the SFV-VA7 vector resulted in effective eradication of both subcutaneous and orthotopic human primary U87MG glioblastoma tumor xenografts without damaging healthy brain tissue in nude BALB/c mice [158]. Improved long-term survival was observed in 16 of 17 treated animals with orthotopic tumors. Remarkably, with the exception of initial transient viremia, none of the mice exhibited neurological symptoms or signs of pathology, therefore demonstrating a high biosafety level for the vector. The oncolytic SFV-VA7 vector was also investigated as a virotherapy candidate for treating unresectable osteosarcoma, the most common primary malignant bone tumor, which typically me-


Table 1.

Zajakina et al.

Investigation of oncolytic replication-competent alphaviral vectors in animal models.

Vector

Tumor model

Administration

Therapeutic efficacy

Refs.

SIN AR339

human ovarian cancer OMC-3

therapeutic (i.p.)

suppression of ascites formation

[153]

SIN AR339

human cervical cancer HeLaS3 and C33A

therapeutic (i.t.)

increase of survival (i.t.), partial tumor reduction

[153]

therapeutic (i.v.)

SFV4

mouse CT26 colon cancer

therapeutic (i.t.)

inhibition of tumor growth, inflammation, increased survival

[155]

SFV-VA7 EGFP, R-Luc

human brain cancer U87

therapeutic (i.v.)

complete tumor reduction (95%)

[159]

SFV-VA7 EGFP

rat glioma BT4C

Therapeutic

ineffective,

[157]

(i.v.), (i.p.), (i.t.)

partial tumor reduction upon i.t. administration

SFV-VA7 EGFP

human lung cancer A549

therapeutic (i.t.)

significant tumor reduction (i.t.)

therapeutic (i.v.)

partial tumor reduction (i.v.)

therapeutic (i.p.)

ineffective (i.p.)

Therapeutic

partial tumor reduction

[156]

[158]

SFV-VA7 EGFP

human melanoma A2058

SIN-GFP

s.c. human glioblastoma U-87 MG

i.t.

local cytopathic effect

[162]

SFV-VA7 EGFP

human osteosarcoma Saos2LM7

therapeutic (i.t.)


[160]

M1 alphavirus

human Hep3B hepatocellular carcinoma, mouse 4T1 mammary carcinoma, mouse B16 melanoma.

i.t.

tumor growth inhibition

[164]

i.v.

mouse ovarian cancer MOSEC

therapeutic (i.p.)

increase of survival,

[161]

SFV + Vaccinia virus

(i.v.), (i.p.), (i.t.)

tastasizes into bones, lungs, and other soft tissues. Subcutaneous human osteosarcoma xenografts were treated with three intratumoral injections of the SFV-VA7-EGFP vector [160]. Treatment with this vector was highly efficient and resulted in a significant reduction of tumor size compared to the oncolytic adenoviral Ad5Δ24 vector. Additionally, highly aggressive orthotopic osteosarcoma xenografts, which are characterized by the invasion of surrounding tissues and the emergence of hematogenous pulmonary metastases, were treated with SFV-VA7-EGFP in a nude mouse model. Intratumoral inoculations of oncolytic SFV significantly enhanced the survival rate in orthotopic osteosarcoma-bearing mice, but no mice were cured. The lack of a curative effect might be associated with an antiviral response because the sera of nude mice treated with SFV-VA7-EGFP contained neutralizing antibodies, primarily IgM, which may have eliminated the virus from peripheral tumors [160]. However, another study demonstrated that the oncolytic SFV vector (wt SFV) had significant therapeutic effects when combined with an oncolytic Vaccinia virus (VV) [161]. In this study, a murine ovarian surface epithelial carcinoma (MOSEC) model was treated with the wt SFV vector using a re-administration strategy with the same or the VV vector. In contrast to the findings for readministering the same virus, heterologous vector inoculation led to remarkably increased oncolysis and generation of an antitumor immune response that significantly prolonged animal survival. Furthermore, infection with SFV encoding a foreign antigen (SFVOVA) followed by infection with VV encoding the same antigen (VV-OVA) enhanced the therapeutic effect generated by virotherapy using oncolytic viruses. Destroying tumor cells by combining

immune response

virotherapy with immunotherapy may lead to broad therapeutic effects due to the dissemination of tumor antigens, resulting in CD8+ control of other ovarian tumor cells that were not infected by the virus. The therapeutic potential of SIN vectors as oncolytic agents has also been examined. The oncolytic properties of the SIN AR339 strain were evaluated for cervical and ovarian cancer therapies [153]. Therapeutic treatment by intratumoral or intravenous injections of the vector resulted in remarkable regression of tumor growth by inducing necrosis in human cervical cancer xenografts (C33A). Histology studies revealed that a single systemic injection of SIN induced necrosis in tumors at a remote site. Moreover, intraperitoneal vector administration in a human ovarian xenograft mouse model significantly suppressed ascite formation, an important therapeutic outcome for treating ovarian cancer. Although SIN AR339 is a replication-competent vector, this strain has not been reported to cause any serious human disease. SIN viruses were also evaluated for the treatment of subcutaneous human glioblastoma xenografts in SCID mice [162]. Several different oncolytic viruses were compared for targeting human glioblastoma, including SIN, VSV, pseudorabies virus, AAV, mouse minute virus, human cytomegalovirus, mouse cytomegalovirus, and simian virus 40. In contrast to other viruses, SIN exhibited tropism in human U-87 MG glioblastoma cells with a strong cytopathic effect upon in vitro infection. Although this study did not determine therapeutic potential, data from an in vivo proof-ofprinciple study substantiated the in vitro results and revealed strong local cytopathic effects in mice treated with intratumoral SIN.


Recently, a new, naturally existing alphavirus M1 was reported as a novel candidate for systemic oncolytic therapy [163, 164]. M1 is a strain of a Getah-like alphavirus that was isolated from culicine mosquitoes collected on Hainan Island in China [165, 166]. M1 preferentially replicated in zinc-finger antiviral protein (ZAP)deficient cancer cells in vitro and in vivo with potent oncolytic efficacy and high tumor tropism. A microarray analysis of patient tissues revealed ZAP-deficiency in 69% of liver, 52% of colon, and 61% of bladder cancers [164]. In animal models, a systemic antitumor effect (tumor growth inhibition) was observed in subcutaneous human Hep3B hepatocellular carcinoma xenografts, orthotopically developed mouse 4T1 mammary carcinomas and mouse B16 melanomas. Importantly, M1-treated mice remained asymptomatic throughout treatment, and there were no obvious differences in body weight between the control and M1-treated groups [164]. Moreover, an assessment of the safety of oncolytic M1 virus infection in adult non-human primates, cynomolgus macaques (Macaca fascicularis) [163] revealed no clinical, biochemical, immunological, or other pathological evidence of toxicity, supporting the potential of M1 for future clinical applications. However, the advantages of M1 over the classic well-characterized oncolytic SFV and SIN vectors remain to be elucidated. In addition to biosafety concerns, the application of oncolytic viruses for cancer treatment is hampered by pre-immunity and strong anti-virus immune responses to repeated vector administration. The immune responses mostly reflect (i) innate immunity via induction of type I IFN signaling, which varies in different tumor types; and (ii) B-cell antibody response against viral structural proteins. Many tumors are defective in type I IFN responses, supporting the therapeutic application of oncolytic vectors. The critical role of type I IFN status was confirmed in several studies of SFV [167] and SIN [168]. IFN-α/β protects the host from local and systemic viral infections such that neighboring or distant uninfected cells also become resistant to virus infection. In one study, IFN-α/β deficient tumors that contained a small (10%) fraction of IFNcompetent tumor cells did not respond to oncolytic SFV treatment in an immunocompetent CT26 colon cancer model [167]. Therefore, a small fraction of virus-resistant cells may determine the outcome of therapy. Moreover, a deficiency in cell IFN-α/βmediated protection alters the biodistribution and tissue tropism of the virus (see section Bio-distribution and tumor targeting). Although alphaviruses are less immunogenic than other oncolytic viruses (e.g., adenoviruses), multiple administrations of the same virus are limited by B-cell antiviral responses, which decreases the therapeutic potential [83]. One strategy for minimizing the effects of the antiviral immune response is the use of different oncolytic vectors for repeated administration. As described above, combining wt SFV and VV had a significant therapeutic effect in mouse ovarian carcinoma [161]. This type of heterologous administration enhanced antitumor effects compared to re-administration of the same virus. Moreover, SFV replication in cancer cells was enhanced by VV infection, which antagonizes the host’s innate antiviral defense (e.g., IFN-α/β responses) [169-171]. However, combined treatment with SFV and VV for malignant glioma was not successful and resulted in reduced targeted replication of vectors in vivo [172]. Although VV is an IFN-insensitive virus, its replication was dramatically inhibited when it was coinjected with SFV. Therefore, broad-spectrum IFN-independent mechanisms of antiviral responses or other escape mechanisms may be involved in brain cancer resistance to oncolytic therapy. Although promising results were obtained for some vector combinations, the combined application of different types of alphaviruses, e.g., SFV and SIN, has not been evaluated. Combining different types of alphaviruses might be a promising alternative in case of absence of cross-reactions of the anti-vector antibody response between closely related alphaviruses.


11

3.2. Expression of Tumor-Associated Antigens (TAAs) Viral vectors show potential for vaccination against cancer because full-length antigen-encoding genes can be efficiently delivered to provide a broader spectrum of potential antigenic epitopes in their native conformation and enhanced immunity via activation of the innate immune response by the virus [173]. Alphaviral vectors are good candidates for developing therapeutic and prophylactic vaccines that express TAAs. Alphaviruses can infect DCs and induce both humoral and cellular immune responses to TAA transgenes through TLR-dependent and independent pathways, resulting in up-regulation of co-stimulatory molecules (CD40, CD80, CD86) and secretion of pro-inflammatory cytokines, including TNF-α, IL-6, IFN-γ, and IL-12 [174-176]. Several TAAs expressed by alphaviruses have been tested in preclinical tumor models (Table 2). The therapeutic effects of SIN vectors were enhanced by vector-dependent transient expression of the TAA model gene LacZ in CT26 colon carcinoma stably expressing β-galactosidase [177]. Although SIN vectors do not target CT26 cells in vivo, a positive therapeutic effect was achieved by targeting vectors in MLNs as a site for early transient heterologous protein expression after intraperitoneal injection. Delivery of TAA into MLNs generated effector and memory CD8+ T cells with robust cytotoxicity against βgalactosidase-positive and β-galactosidase-negative tumor cells. Interestingly, these results indicated that SIN-LacZ therapy for CT26 β-galactosidase-producing tumors induced epitope spreading, which led to the development of immunity against other antigens expressed on the CT26 tumors. Importantly, for the first time, a four-step model was proposed for the activation of CD8+ T cells during TAA therapy: Step 1: TAA expression in the MLNs and activation of T cells and NK cells. Step 2: destruction of tumor cells stimulated by TAA-specific CD8+ cytotoxic T cells, resulting in the release of TAAs. Step 3: capture of these antigens by APCs and their presentation to CD8+ T cells in tumor-draining lymph nodes, resulting in epitope spreading and the induction of CD8+ T cells specific for other antigens in heterogeneous cancer cell populations. Step 4: generation of memory CD8+ T cells against a variety of TAAs. Therefore, the therapeutic benefit from SIN-TAA does not require direct targeting of tumor cells, but the induction of antitumor immunity is essential for efficient therapy. Moreover, the spread of epitopes observed for SIN vectors may be crucial for systemic and stable therapeutic effects. Melanoma Antigens VEE vectors have been used for TAA-based therapy by expressing melanoma antigens in preclinical models. Melanoma is one of the most aggressive forms of skin cancer. Melanoma tumors develop from melanocytes and express specific TAAs that can be categorized as differentiation antigens, such as p75/tyrosinaserelated protein TRP-1, Pmel17/gp100, MART-1/Melan-A and the retained intron in tyrosinase-related protein (TRP-2-INT2), as well as TAAs such as melanoma cell adhesion molecules (MUC18) or MAGE [178]. These antigens are ideal targets for melanoma immunotherapy because they are preferentially expressed in melanoma cells and melanocytes. Suboptimal results were obtained using the TRP-2 (transmembrane melanosomal glycoprotein) TAA as a therapeutic gene to control melanoma growth [179]. VEETRP-2 VLPs induced long-term tumor protection when administered as late as 5 days after tumor inoculation in a mouse B16 melanoma model. Importantly, vaccination with VEE-TRP-2 was more effective than the combination of VEE-Tyr and VEE-gp100 vectors. Furthermore, the efficiency of combined treatment with all three VEE vectors was not significantly improved compared to the VEE-TRP2 vector alone. Another study demonstrated the high prophylactic potential of VEE VLPs encoding human or mouse TRP-1 for inducing immune responses and delaying tumor growth in immunocompetent


Table 2.

Zajakina et al.

Tumor associated antigens (TAAs) expressed by alphaviral vectors.

TAA

Tumor Model

Vector

Administration

Therapeutic Efficacy

Refs.

LacZ model antigen

CT26 colon carcinoma expressing β-gal (i.p. and s.c. models)

SIN VLP

i.p.

CD8 T cell response,

[177]

TRP-2

mouse melanoma B16

VEE VLP

s.c. prophylactic


[179]

TRP-1

mouse melanoma B16

VEE VLP

s.c. prophylactic

immune response, partial tumor reduction

[180]

TRP-1

mouse melanoma B16

SIN DNA

i.m. prophylactic

immunity, tumor prevention (60-70%)

[183]

MUC18

mouse melanoma B16 transduced with MUC18

SIN DNA

prophylactic (s.c.)

immune response, tumor prevention (50%)

[184]

Her/neu

human breast cancer NT2

VEE VLP + DCs

s.c. (VRP-DCs injected in the right axillary mammary gland adjacent to established tumors)

prophylactic immune response, partial tumor growth inhibition

[197]

rat breast cancer 13762MAT-B-III

VEE VLP

s.c.


[189]

mouse breast cancer model A2L2

SIN DNA

footpad injection

Ineffective

[188]


SIN DNA

prophylactic (i.m.)

tumor prevention (50%), reduction of metastasis, partial tumor reduction

[190]


SIN DNA

prophylactic (i.m.) (i.d.)

tumor prevention (80%), reduction of metastasis, increased survival

[193]

HPV16 E6 E7

mouse cervical cancer model TC-1

SFVenh VLP

prophylactic (s.c.), (i.p.)

complete tumor prevention (100%)

[202]

Sig/E7/

mouse cervical cancer model TC1

SIN RNA

prophylactic (i.m.)

immune response, reduction of metastasis

[208]


SIN VLP

prophylactic (i.m.)

complete tumor prevention (prophylactic settings), reduction of pulmonary nodules (therapeutic settings), immune response

[209]

therapeutic (i.m.)

HPV16 E7

mouse cervical cancer C3

VEE VLP

prophylactic (s.c.)


[210]

HPV16 E6, E7

cervical cancer model HLF16

VEE VLP

prophylactic (s.c.)


[211]

HPV16 E6, E7

mouse cervical cancer C3, TC1

VEE VLP

prophylactic (s.c.)


[212]

PSCA

mouse prostate cancer TRAMPC-2

VEE VLP +cDNA (PSCA)

s.c. prophylactic

tumor prevention (76%)

[219]

STEAP

mouse prostate cancer TRAMPC-2

VEE VLP

s.c. prophylactic/therapeutic

partial tumor prevention, increased survival

[218]

PSA

transgenic DR2bxPSA F1 mouse model

TC-83 repliconderived VEE VLPs

i.m.

immune response, elimination of PSA-expressing cancer cells

[216]

LAMP-1 calreticulin/E7

inhibition of tumor growth

melanoma-bearing mice [180]. This study used a promising heterologous prime/boost immunization regimen to avoid antivectorneutralizing immunity effects. This type of vaccination involves initial (prime) and subsequent (boost) immunizations with different vectors. Immunization with plasmid DNA encoding murine or human TRP-1 was directly compared to VEE VLPs encoding the

same antigens. In contrast to plasmid DNA vaccination, which requires xenogeneic (human) tyrosinase to overcome immunologic ignorance and/or tolerance, alphavirus constructs containing either syngeneic (mouse) or human tyrosinase induced antibody and T cell responses and ensured tumor protection when administered either alone or with the plasmid DNA prime/VLPs boost approach. It is


not clear why the VEE VLPs, in contrast to plasmid DNA, activated an immune response to the syngeneic antigen in the B16 melanoma mouse model. The authors suggested that viral replication may be involved in breaking immune tolerance. Specifically, RNA viruses, which replicate through dsRNA intermediates, can trigger TLR3dependent synthesis of type I IFNs, which function as a broad stimulus of the immune response [128]. Errors generated during viral RNA replication and defective ribosomal products (DRiPs), including premature truncated or misfolded polypeptides, might be an additional heterogenic source of peptides for MHC I processing [181, 182]. Both the high levels of viral protein expression and cellular stress induced by viral infection can alter processing and enhance the presentation of full-length antigens. Advances in targeted prophylactic and therapeutic immunotherapy for melanoma have also been demonstrated in preclinical studies of other alphavirus vectors. The efficacy of the DNA-based SIN vaccines pSIN-mTRP-1 and SIN-hTRP-1 was investigated in a mouse melanoma B16 model [183]. This study was one of the first demonstrations of prophylactic immunization with an alphavirus DNA vector. Intramuscular injection of plasmid DNA was able to break immunological tolerance and induce immunity against melanoma when administered 5 days prior to cancer cell challenge. MUC18 is another melanoma antigen that appears in late primary and metastatic melanoma but not in healthy melanocytes. The SINCP plasmid DNA vector was used to express the MCAM/ MUC18 murine melanoma cell adhesion molecule to induce immunity against murine melanoma [184]. Application of this vector showed no antitumor effect against mouse B16F10 cells, possibly due to the extremely low expression of murine MUC18 in these melanoma cells. To increase MUC18 antigen expression in tumor cells, new B16F10 cells transduced with MUC18 were obtained. In this case, vaccination against MUC18 resulted in the induction of humoral and CD8+ T cell immune responses. To investigate the efficacy of recombinant alphavirus-based vaccines for stimulating human immune responses, SFV VLPs encoding MAGE-3 were administered in humanized BALB/c mice (Trimera murine model) [185]. The SFV vector elicited human MAGE-specific humoral and CTL responses in the Trimera mouse model. Immunization with plasmid DNA encoding TAAs has several advantages compared to other vaccination strategies, such as peptides and VLPs. First, plasmid DNA isolated from Escherichia coli contains unmethylated immunostimulatory CpG sequences that may act as potent immunologic adjuvants via TLR9-dependent signaling [186]. Second, DNA is simpler to produce and purify in large quantities compared to proteins or viral vectors. However, DNA vaccines alone have not been sufficient for inducing complete or stable immunity and have exhibited suboptimal efficacy in human and nonhuman primates (for a review see [187]). Breast Cancer Antigens The efficiency of DNA vectors greatly depends on the TAA and tumor model of choice. Several early studies reported the application of SIN DNA vectors and VEE replicon particles for therapeutic and prophylactic vaccination of breast cancer models with Her2/neu TAA [188-190]. The Her2/neu tumor antigen is a member of the tyrosine kinase receptor family that is overexpressed in 30-40% of breast cancer cells and is correlated with increased metastasis and poor prognosis due to high mitotic activity, mutation of the p53 tumor suppressor gene, absence of bcl-2 and negative estrogen receptor status [191, 192]. A DNA-based SIN vector expressing the neu gene, ELVIS-neu, was used for prophylactic intramuscular immunization 14 days before injection of cancer cells overexpressing neu [193]. Strong protection of mice against tumor challenge was observed in this study. Vaccination led to a reduction of the incidence of lung metastasis from the orthotopic mammary fat pad tumors and reduced the number of lung metastases resulting from intravenous injection of neu-overexpressing mouse breast cancer


13

A2L2 cells. Importantly, intradermal vaccination also provided tumor protection and required 80% less plasmid to obtain a similar level of protection. Wang and colleagues confirmed the beneficial results of therapeutic/prophylactic cancer vaccines based on the plasmid DNA SIN neu vector for treating pre-existing tumors [190]. The therapeutic efficacy of the pSINCP/neu vaccine depended on the order of vector and cancer cell injection, which indicated that the prophylactic vaccine was effective only when administered before tumor challenge. However, the sequential administration of neu-expressing SIN DNA vectors and adenovirus particles in a prime-boost protocol was therapeutically effective (prolonged the overall survival rate) when tumor cells were injected intravenously before vaccination. Engineered viral vectors can efficiently deliver tumor antigens directly to DCs in the context of an immunostimulatory viral infection, resulting in optimal DC activation [194, 195]. VEE VLPs efficiently transduce human DCs, resulting in maturation and secretion of proinflammatory cytokines in vitro [196]. The VEE-DC vaccine was immunogenic in vivo and was capable of inducing both cellular and humoral immunity against neu [197]. VEE/neu transduction of DCs resulted in phenotypic maturation and secretion of the proinflammatory cytokines TNF-α and IL-6 but not the immunosuppressive cytokine IL-10. VEE-infected DCs also secreted large amounts of type I IFNs, which participated in the activation of NK cells and enhanced the cross-presentation of antigens to CD8+ T cells, in addition to enhancing the clonal expansion of antigen-specific CD4+ and CD8+ T cells. Therefore, a positive effect for destroying the immunosuppressive tumor microenvironment was achieved. However, although several studies observed partial tumor protection and growth inhibition, the efficiency of alphaviral Her2/neu TAA-based vaccines for interrupting immunotolerance as a therapeutic vaccine was suboptimal, and additional significant progress in this area has not been demonstrated. Cervical Cancer Antigens Human papilloma virus (HPV) proteins have been identified as cervical cancer-associated antigens [198]. The HPV E6 and E7 oncogenes inhibit tumor suppressor genes and are widely used targets for developing cervical cancer vaccines. In early research, SFV VLPs expressing the HPV-16 E6 and E7 as separate proteins derived from the most prevalent HPV type, HPV type 16, were tested as prophylactic vaccines in a dose-dependent manner in a mouse TC-1 cervical cancer model expressing HPV16 E6E7 [199]. Preimmunization with three injections of 104 pSFV-E6E7 VLPs induced an HPV-specific CTL response in 50% of the mice, whereas three inoculations with an increased virus dose of 106 resulted in a CTL response in all treated animals. Furthermore, prophylactic immunization with an increased dose of 5 × 106 of SFV-E6E7 VLPs protected 40% of the mice from tumor challenge, whereas lower doses were less efficient. To improve the cellular immune responses against E6 and E7, a new vector encoding a fusion protein of E6 and E7 together with the SFV core translational enhancer (pSFV3enh-E6,7) was generated [200]. The SFV core translational enhancer significantly increased the expression of the E6,7 fusion protein. Immunizations with 5 × 106 SFV3-enhE6 and 7 VLPs protected four out of five mice from developing tumors, and a second tumor challenge in protected tumor-free animals revealed complete long-term protection against tumor occurrence. In addition to prophylactic vaccination, a therapeutic treatment strategy against HPV-induced cancer was developed based on recombinant SFV particles encoding a fusion protein of E6 and E7 [201]. Immunization with SFV3enhE6-7 particles induced strong HPV-specific CTL activity and eradicated established TC-1 HPVtransformed tumors. Subcutaneous injections of the vector on days 2, 7, and 14 after TC-1 cell challenge led to rapid CTL response induction and efficient protection against tumor growth. Remarka-


bly, 90-100% of treated mice were protected after a second tumor challenge. Another study confirmed the high potential of the proposed SFV-enhE6,7 VLP vaccine via intravenous and intramuscular administrations [202]. The authors concluded that the route of immunization strongly influences the dose of viral vector needed to induce the CTL response and the therapeutic effect. As few as 5x104 SFVenhE6-7 primed and boosted by intravenous injection were sufficient to eradicate tumors in six out of seven treated mice. Interestingly, an adenovirus Ad5 type vector that expressed the same antigen construct (Ad-eE6,7) had a lower prime-boost CTL response and therapeutic efficacy than SFVeE6,7 [203]. Moreover, for the delivery mode, an intradermal injection of SFVeE6,7 particles via tattoo injection resulted in levels of immune response that were higher than or similar to those of intramuscular injections [204]. Tattoo injections may be an effective and simple administration method for the induction of potent immune responses by alphavirus-based vector immunotherapy. To prolong expression and enhance the immune response against the E7 antigen, a plasmid DNA-based SFV vector, pSCA1, encoding E7 fused with BCL-xL was generated [205]. BCL-xL is a member of the BCL-2 family of anti-apoptotic proteins. DCs transfected with this construct exhibited delayed cell death, generated significantly higher E7-specific CD8(+) T cell-mediated immune responses and had better antitumor effects than pSCA1 encoding the wild-type E7 gene in vaccinated mice. The anti-apoptotic function of BCL-xL was important for enhancing antigen-specific CD8 + T cell responses in vaccinated mice because a point mutant of BCLxL that lacked anti-apoptotic function was ineffective. Therefore, the use of anti-apoptotic proteins to delay suicidal DNAinduced cell death may enhance the potency of suicidal DNA vectors. In summary, the SFV-enhE6,7 construct represents a safe and efficient vaccine for cervical cancer and induces a very strong, long-lasting CTL response that is sufficiently strong to eradicate existing tumors and prevent tumor development in a prophylactic setting. In addition to VLPs and plasmid DNA vaccines, alphaviral naked RNA replicons have the potential to be used for immunization. Similar to infection with VLPs, RNA transfection eventually causes transgene expression, followed by lysis of transfected cells due to the cytopathic effect of viral RNA replication. Moreover, RNA immunizations obviate concerns about integration into the host genome, a significant advantage over plasmid DNA vaccines. SIN self-replicating RNA vectors (SINrep5) were developed to induce E7-specific immunity in a TC-1 murine model [206]. Intramuscular inoculation of RNA encoding the HPV E7 oncogene alone (SINrep5-E7) induced poor humoral and cellular immune responses and provided no protection against tumor challenge. However, the SIN RNA replicon containing an E7 fusion with the Mycobacterium tuberculosis heat shock protein 70 (HSP70) gene generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than vaccines encoding the wild-type E7 gene. In vitro studies demonstrated that E7 from the E7/HSP70 construct was processed by DCs and presented more efficiently through the MHC class I pathway than wild-type E7 protein. Furthermore, another construct that expressed the E7 protein as a fusion with a secreted Sig protein and lysosome-associated membrane protein-1 (LAMP-1) in the SINrep5-Sig/E7/LAMP-1 vector resulted in E7-specific CD4+ helper T cell and CD8+ cytotoxic T cell activity and increased in vivo antitumor effects [207]. Fusion of the Sig/LAMP-1 endosomal/lysosomal sorting signal with the E7 protein significantly enhanced oncoprotein processing and targeting to the endosomal and lysosomal compartments for an overall enhancement of the efficiency of antigen presentation through the uptake of apoptotic cells by APCs at sites of vector RNA inoculation. These results indicated that fusing E7 with immune stimulating sequences support efficient antigen presentation and may

Zajakina et al.

greatly enhance the potency of self-replicating RNA vaccines. Moreover, the intracellular delivery of naked alphaviral RNA can be enhanced by standard non-viral delivery approaches (e.g., by cationic lipids) or cell-penetrating peptides, as demonstrated for SFV RNA [208]. Cheng and colleagues investigated the efficacy of prophylactic and therapeutic vaccines based on SIN VLPs expressing both E7 and calreticulin (CRT) [209]. CRT is an ER Ca2+-binding transporter that participates in antigen processing and presents with MHC class I. The generated SINrep5-CRT/E7 VLP vector induced E7-specific immune responses, exhibited an anti-angiogenic effect, and provided strong antitumor activity in the mouse TC-1 model. Intramuscular immunization of mice with SINrep5-CRT/E7 VLPs one week prior to challenge with TC-1 cells ensured excellent protection of all treated animals. To test the vector’s efficiency in therapeutic settings, SINrep5-CRT/E7 VLPs were intramuscularly inoculated into immunocompetent and nude mice two days after tumor cell injection. Although this strategy did not result in complete tumor eradication as a prophylactic approach, a significantly lower number of pulmonary tumor nodules were observed in both mice groups after intravenous challenge with TC-1 cells. VEE VLP-based vectors expressing E7 were also tested as candidates for developing cervical cancer vaccines. Two subcutaneous pre-immunizations with VEE-E7 VLPs two weeks prior to cancer cell injections prevented tumor formation in mouse C3 cervical cancer models [210]. Moreover, mice challenged three months after the first cancer cell injection did not develop tumors, indicating induction of long-term memory responses by the vector. However, further studies demonstrated that the therapeutic treatment of established tumors was efficient in only 67% of treated mice. The efficacy of the vaccine based on the VEE vector was enhanced by bicistronic IRES-based expression of both E6 and E7 antigens from the same vector [211, 212]. To test the HLArestricted capabilities of the vaccine, HLA-A0201 transgenic mice were used to establish an HPV-sensitive tumor model. Heart and lung tissue-derived fibroblasts isolated from HLA-A*0201 mice were transfected with HPV16 E6, E7 and H-Ras V12 oncogenes to generate a tumorigenic cell line for the HLA-A0201 model. Preimmunization of mice with VEE-E6E7 VLPs induced specific T cell immune responses against HLA-A0201-restricted HPV-16 epitopes and protected 100% of mice from tumor challenge. As a therapeutic immunization strategy, when the VEE VLPs were inoculated after tumor challenge, the treatment did not completely eradicate the tumors. However, therapeutic vaccination significantly reduced tumor growth in transgenic mice. Prostate Cancer Antigens VEE vectors were used for immune stimulation against specific prostate cancer antigens, including the prostate-specific membrane antigen (PSMA), the six transmembrane epithelial antigens of the prostate (STEAP), and the PSCA [213]. The VEE VLP vector, which expresses human PSMA, demonstrated strong humoral and cellular immunity in mice upon subcutaneous administration [214]. Due to the absence of relevant PSMA tumor challenge models, clinical trials have examined the efficacy of VEE-PSMA VLPs [215]. Moreover, PSA-specific cellular and humoral immune responses were recently investigated in an HLA-DR transgenic mouse model [216]. PSA-encoding VEE VLPs derived from the TC-83 replicon overcame tolerance to the “self” antigen and induced strong PSA-specific CD8+ T cell and Th1-type antibody responses. Preventive vaccination with VLPs-PSA resulted in the elimination of PSA-expressing tumor cells and significantly inhibited tumor growth in a stringent mouse model of prostate cancer in DR2bxPSA F1 mice. The STEAP antigen is also an attractive target for vaccine development. STEAP is predominantly expressed in prostate tissue and is up-regulated in many prostate cancer cell lines [217]. The


prophylactic potential of VEE VLPs expressing mouse STEAP was assessed in TRAMPC-2 prostate tumor-bearing mice preimmunized with VEE-STEAP VLPs. A specific STEAP induced immune response and significantly prolonged overall survival was observed in the mice. The therapeutic effect of the VEE vector was evaluated by co-administration of plasmid DNA encoding the STEAP gene, which resulted in a short but statistically significant delay of tumor growth. Positive results were also obtained using PSCA as a target. Similar to STEAP, the PSCA antigen is upregulated in a large proportion of localized and metastatic prostate cancers. Prophylactic pre-immunization of transgenic TRAMP mice with the PSCA encoding DNA plasmid followed by VEE-PSCA VLP administration elicited a specific anti-PSCA immune response and tumor protection in 90% of TRAMP mice [218, 219]. Therefore, early studies and recent developments have demonstrated that alphaviral vectors are attractive tools for expressing different types of TAAs. Moreover, cell death induced by virus replication can facilitate the uptake of apoptotic and necrotic cells and cell fragments by DCs and other antigen-presenting cells, leading to enhanced cross-presentation of expressed heterologous TAA and other additional TAAs derived from destroyed cancer cells. 3.3. Expression of Immunomodulating Cytokines The modest level of immune responses and the requirement for multiple immunizations suggest that the action of alphaviral vectors could be enhanced by immune stimulating cytokines. Cytokines are native immune cell-produced molecules that can be used to directly stimulate immune cells and enhance tumor cell recognition by cytotoxic effector cells. Numerous studies have demonstrated that cytokines have broad anti-tumor activity, and many cytokine-based approaches have been employed for cancer therapy. Several cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18 and IL-21, have been evaluated in clinical trials. The anti-tumor effects of several Table 3.


types of interleukins expressed by alphaviral vectors have been described in a variety of mouse tumor models (Table 3). Interleukin-12 (IL-12) has been expressed by SFV and SIN vectors to overcome the immunosuppressive environment in tumors. IL-12 is a multifunctional cytokine that activates innate and adaptive immune responses, facilitates the presentation of tumor antigens through the up-regulation of class I and II MHC molecules, triggers CD4+ T cells to differentiate into type 1 T helper (Th1) cells, induces IFN-γ production and subsequently programs the tumor microenvironment [220]. These cascades lead to cytotoxic immune responses in tumors. SFV vectors expressing IL-12 induced therapeutic anti-tumor effects mediated by activation of CTLs in mouse lung carcinoma, melanoma, woodchuck hepatocellular carcinoma, colon cancer and mastocytoma models. For example, the expression of cytokine genes induced tumor-specific immunity and dramatically inhibited tumor cell growth in a melanoma model. A single intratumoral injection of recombinant SFV VLPs expressing IL-12 caused dramatic tumor necrosis and resulted in 70-90% inhibition of tumor growth in a B16 mouse melanoma model [221]. However, complete tumor regression was not achieved in this early study. Several SFV vectors have been engineered to express different types of cytokines to activate antitumor immunity in colon cancer models. A single intratumoral injection of 107 and 108 SFV VLPs expressing IL-12 resulted in complete tumor regression in 36% and 80% mice, respectively, in an MC38 colon adenocarcinoma model [83]. Moreover, fusion of IL-12 with a natural SFV capsid translation enhancer significantly increased IL-12 expression and led to more efficient tumor regression in treated mice. Six doses of hightiter SFV10-Enh VLPs expressing IL-12 was necessary for complete tumor regression in all colon carcinoma-bearing mice (MC38 model). During the treatment stage, tumor swelling due to intratumoral necrosis and inflammation and intratumoral influx of CD4 +

Immunomodulating cytokines expressed by alphaviral vectors.

Cytokine

Tumor model

Vector

Administration type


Refs.

IL-12

mouse melanoma B16

SFV VLP

i.t.


[221]

(70%-90%) mouse colon cancer MC38

SFV VLPs

i.t.

complete tumor reduction (92%)

[83]

mouse colon cancer CT26;

SFV-E VLPs

i.t.

complete tumor reduction

[223]

spontaneous hepatocarcinoma in LPK/c-myc transgenic mice

SFV VLPs

i.t.

tumor growth arrest, 100% survival rate

[227]

woodchucks liver cancer

SFV-E VLP

i.t.


[226]

human ovarian cancer ES2

Sin VLPs

i.p.


[230]

IL18

mouse colon cancer CT26

SFV VLPs

i.t.

partial tumor reduction, complete tumor reduction (36%)

[232]

IL-18


SFV-E VLP

i.t.

complete tumor

[232]

mouse 4T1 breast cancer

WCH17 IL-15,

15

IL-12

reduction (33%) IFNalpha


SFV VLPs

i.t.

eradication of 58% of established tumors

[233]

GM-CSF

mouse ovarian cancer MOT

SFV VLPs

i.p.

increased survival, partial tumor reduction, immune response

[235]


and CD8+ T cells and other immune cells was observed, which promoted the anti-tumor effect [222, 223]. Chikkanna-Gowda and colleagues observed promising results in the 4T1 mouse breast cancer model [223]. In this study, intratumoral administration of high-titer SFV-Enh-IL-12 VLPs led to complete tumor regression in four out of six mice and significantly reduced the amount of lung metastases. In addition to direct intratumoral expression of immunomodulating cytokines, therapeutic immunization with modified DCs that have been pulsed with SFV IL-12 significantly prolonged the survival of mice with brain tumors of the B16 cell line [224]. A good survival rate was also observed after stimulating the immune system with DCs transduced with SFV-IL-18 VLPs in combination with systemically administered IL-12 in the same B16 brain tumor model [225]. In this study, local delivery of DCs expressing IL-18 and systemic injection of IL-12 enhanced the induction of the Th1 response and tumor-specific CD8+ T cell responses. Moreover, the use of T cell-depleted mice and IFN-γ-neutralized mice demonstrated the important role of IFN-γ and CD8+ T cells in IL-18mediated protective immunity. In preclinical hepatocellular carcinoma studies, woodchucks chronically infected with woodchuck hepatitis virus serve as a model for liver cancer therapy development. The therapeutic potential of SFV-E-IL12 VLPs was evaluated in woodchucks bearing liver tumors [226]. A single intratumoral injection of SFV-E-IL12 VLPs provided partial but vector dose-dependent tumor regression in 58% of the treated animals, leading to a reduction of tumor volume of up to 70% within 4 weeks after treatment. This promising therapeutic outcome was associated with the activation of immune responses against hepatocellular carcinoma and woodchuck hepatitis virus viremia. However, tumor growth was subsequently restored. Recently, intratumoral inoculation of SFV-IL-12 VLPs was compared with hydrodynamic injections of plasmid DNA pTonL2(T)-mIL12, which permits liver-specific and inducible IL12 expression, in L-PK/c-myc transgenic mice with spontaneous hepatic tumors [227]. SFV-IL-12-induced growth arrest was observed in most tumors, with a 100% survival rate, in contrast to plasmid-treated mice, in which tumor arrest was less evident and the survival rate was slightly lower, despite higher and more sustained levels of IL-12 and IFN-γ in serum. Virus-mediated induction of the type-I IFN response in infected tumors explains why the short-term SFV-dependent IL-12 expression was sufficient to mediate anti-tumor responses. This conclusion was confirmed in a recent study that demonstrated that antitumor efficacy resulted from the interplay between SFV-produced IL-12 as a transgene and alphavirus-mediated induction of IFN-α/β acting as endogenous mediators that signal to hematopoietic cells and lead to a remarkably stronger CTL response against tumor cells [228]. Although the exact mechanism of this interaction remains unclear, the type I IFN response induced by SFV-IL12 was dependent on IPS-1- and TRIF-mediated pathways, which are important for stimulating innate immune responses. By contrast, the host immune response, specifically, the antiviral type I IFN response limits alphavirus replication and thus reduces the efficacy of oncolysis and the related bystander effect. Tumors that are defective in IFNα/β are more susceptible to SIN infections in vitro and in vivo, which enhance vector tropism in cancer cells and increase the efficacy of oncolysis [229]. Therefore, type I IFN status, which varies in different tumor types, is an important factor in alphaviral vectorbased cancer immunotherapy. Incorporating IL-12 genes into the SIN vector enhanced the efficacy of therapy in the human ovarian cancer ES-2 model [151, 230]. The anticancer efficacy of the SIN IL-12 vector is largely dependent on NK cells because depleting NK cells caused a significant decrease in vector therapeutic potential for ovarian cancer-

Zajakina et al.

bearing C.B-17-SCID beige mice with selective impairment of NK cell function [231]. In addition to IL-12, other IFN-γ-inducing cytokines have been expressed by alphaviral replicons. IL-15 and IL-18 can potentiate anti-tumor immune responses for SFV and SIN vectors in mouse ovarian and colon cancer models [232], resulting in partial tumor reduction upon VLP administration. For example, colon carcinoma tumor-bearing mice were treated with six inoculations of high-titer SFV10-E VLPs expressing the murine IL-18 gene carrying an IgGkappa leader sequence [233]. In addition to anti-angiogenic activity, IL-18 is an IFN-γ-inducing cytokine that has a key role in the early activation of the Th1 cell-mediated immune response. Although delayed growth of all treated tumors was observed, complete tumor regression was detected in only 33% of treated mice, with activation of avascular and suppurative necrosis. In another study, an SFV vector expressing murine IFN-α was evaluated in TC-1 tumor-bearing mice expressing E6 and E7 proteins of human papillomavirus [234]. IFN-α is a pleiotropic cytokine that has direct anti-proliferative activity as part of the innate immune response by enhancing antitumor immunity [234]. Although IFN-α has antiviral activity, SFV-derived IFN-α was produced in cancer cells at high levels. Intratumoral injection of the SFV-IFN-α vector into subcutaneous TC-1 tumors induced an E7specific CTL response and modified the tumor microenvironment by reducing the percentage of Tregs and activating myeloid cells. Consequently, SFV-IFN-α eradicated 58% of established tumors treated 21 days after cancer cell implantation. Moreover, long-term tumor-free survival and very low vector toxicity were observed. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an immunostimulatory cytokine that activates DCs and can reprogram the tumor microenvironment. SFV VLP vectors were utilized for ovarian cancer immunotherapy with GM-CSF [235]. Intraperitoneal injection of SFV-GM-CSF VLPs in a murine ovarian tumor model increased the number of infiltrated macrophages and neutrophils. Modest tumor growth inhibition with no survival benefit was observed. High doses of SFV-derived GM-CSF might cause detrimental effects via immune suppression by activating and expanding myeloid-derived suppressor cells (MDSCs) [236]. The delivery of cytokines by viral vectors remains a promising alternative to the administration of cytokines as recombinant proteins because the vectors ensure intratumoral expression of cytokines and a slow increase in serum levels, thereby avoiding cytokine toxicity and instability. The combination of alphavirus-based cytokine therapy with other treatment strategies is described below. 3.4. Combining Anti-Cancer Therapeutic Strategies The immunosuppressive effects of the tumor microenvironment and tumor-escape mechanisms may explain the failure of IL-12based immunotherapy for human cancers [237]. However, the combination of different therapeutic strategies has demonstrated promising results in preclinical models (Table 4). These novel approaches may help overcome the obstacles that prevent alphavirus deployment for broader clinical applications. Combining chemotherapy agents with agents that enhance effector T cell functions and agents that inhibit immunosuppressive elements, such as MDSC, Tregs, and M2 macrophages, to restore impaired immunity in cancer may be complementary and possibly synergistic [238]. TAAs + cytokines The use of immunostimulatory cytokines to enhance immune responses against TAA is one strategy for developing advanced cancer vaccines. Recently, an adjuvant effect of IL-12 was demonstrated for combined administration of IL-12 and CEA, which were both expressed by VEE replicon particles in mice harboring CEAexpressing MC38 colon cancer cells [147]. Vaccination with VEE/CEA plus VEE/IL-12 was superior to VEE/CEA or VEE/IL12 alone for inducing antitumor activity and prolonging survival in


Table 4.


17

Combined cancer treatment modalities in presence of alphaviral vectors.

Combined treatment

Tumor model

Vector

Administration type


Refs.

IL-12 + CEA

CEA-expressing colon cancer MC38-CEA-2 cells

VEE-IL12 VLPs

footpad injections

enhanced CEA-specific immune responses and survival prolongation in tumor-bearing mice

[147]

B16F10 mouse melanoma cells, stably expressing human survivin and hCGβ

SFV DNA immunisation

i.m. electric pulse

induction of protective immune response, increase of survival

[241]

mouse cervical cancer model TC1

SFV-E VLP +

Therapeutic (s.c.)

complete tumor

[242]

MC38 mouse colon cancer and B16-OVA melanoma

SFV/IL-12 VLPs

mouse B16 melanomas (B16-OVA and B16.F10) and mouse TC-1 lung carcinoma

SFV/IL-12 VLPs

TRP2 + anti-CTLA-4 m Ab, or anti-GITR mAb

mouse B16 melanoma

VEE/TRP2 VLPs

HPV16 E6, 7


SFV3-enhE6,7 +SFVIL12 VLPs

mouse A2L2 breast cancer model

GM-CSF + TAA: human chorionic gonadotropin (hCGβ) + survivin (SUR) HPV16 E6, 7 + IL-12 IL-12 + anti-PD-1 mAb

IL-12 + agonist CD137 mAb

+ IL-12 Her2/neu + doxorubicin paclitaxel

Sin/LacZ + Paclitaxel SFVeE6,7 + Sunitinib

VEE-CEA(6D) VLPs

SFV VLP

reduction (28%) i.t. (SFV/IL-12)

tumor regression and prolonged survival

[253]

tumor eradication, prolonged survival

[255]

tumor regression, enhancemt of anti TRP-2 immunity

[246]

s.c. (SFV3-enhE6,7)

complete tumor

[242]

s.c.(SFV/IL12)

reduction (28%)

SinCP/neu DNA

i.m. (SinCP/neu DNA)

enhanced immunity, tumor growth inhibition

[188]

VEE/neu VLPs

i.m. (VEE/neu VLP)

SCID mice bearing human ovary ES-2/Fluc tumors

Sin/LacZ VLPs

i.p. Sin/LacZ VLPs

inhibition of tumor growth, increased survival

[151]


SFVeE6,7 VLPs

i.m. (SFVeE6,7)

inhibition of tumor growth, increased survival

[257]

100% tumor-free survival

[258]

s.c. and i.t.

Inefficient

[249]

s.c. and i.t.

Enhanced

[249]

i.p. (antiPD-1 Ab) i.t. (SFV/IL-12) i.p. (agonist CD137 Ab) s.c. (VEE/TRP2) i.p. (Ab)

i.p. (sunitinib) SFVeE6,7 + Sunitinib + tumor irradiation VEGFR-2 + IL-12


SFVeE6,7 VLPs

i.m. (SFVeE6,7) i.p. (sunitinib) local tumor irradiation


SFV/VEGFR-2 VLPs SFV/IL-12 VLPs

VEGFR-2 + IL-4


SFV/VEGFR-2 VLPs

survival, significant tumor regression

SFV/IL-4 VLPs VEGFR-2 + IL-12

mouse melanoma

SFV DNA immunization

i.m. electric pulse

induction of protective immune response, increase of survival

[250]

metastatic 4T1 mouse breast cancer

SFV/IL-12 VLPs

i.t.

inhibition of metastasis, longterm survival

[264]

B16

+ chorionic gonadotropin+ surviving Salmonella typhimurium (LVR01) + IL-12


tumor-bearing mice. Moreover, local injection of VEE/IL-12 at the VEE/CEA injection site provided more potent activations of CEAspecific immune responses than injection of VEE/IL-12 at a distant site. Co-injection of VEE/IL-12 was more efficient than applying recombinant IL-12 to stimulate CTL induction by VEE/CEAinfected DCs at the lymph nodes because local IL-12 expression from the VEE vector produced a longer paracrine effect. In another study, GM-CSF was applied via the SFV DNA vector as an adjuvant for a TAA vaccine to express survivin [239] and chorionic gonadotropin [240] fusion proteins. This SFV replicon-based DNA vaccine was effective against pre-existing B16 mouse melanoma tumors expressing the same TAAs [241]. Moreover, the key role of GM-CSF in the induction of protective immune response was demonstrated. The therapeutic efficacy of the SFV3-enhE6,7 VLP-based vaccine expressing HPV E6,7 fusion protein was improved by coinoculation with different doses of SFV-IL-12 VLPs [242]. The results of co-administration of both vectors significantly depended on the viral dose and injection regimen in mouse TC1 cervical cancer models. Synergistic antitumor effects were observed only at a low dose of SFV-IL12. Although the mechanism of the doseresponse effect for SFV-IL12 is not clear, specific attention should be given to determining the optimal adjuvant dose in combined treatments. Furthermore, heterologous vector prime-boost immunization was advantageous compared to a single immunization strategy [243]. A heterologous prime boost with SFV3-enhE6,7 VLPs and virosomes containing the E7 protein resulted in higher numbers of antigen-specific CTLs in mice than homologous protocols with only VLPs or virosomes. However, the high number of CTLs initially primed by the heterologous protocols did not correlate with enhanced functional antitumor responses in vivo. Another study assessed combined vaccination with a plasmid DNA-based SIN vector expressing human gp100 melanoma antigen and murine IL-18 for therapy in a B16-gp100-implanted brain tumor model [244]. Three prophylactic pre-immunizations with both pSIN-hgp100 and pSIN-IL-18 DNA constructs induced a specific antitumor CTL immune response and prevented tumor formation. By contrast, therapeutic vaccinations of mice with B16-hgp100 tumors significantly prolonged animal survival. For the combined treatment, the median survival rate was 90 days, compared to 24-28 days for mice treated with either pSIN-hgp100 or pSIN-IL-18 DNA. As mentioned above, alphaviruses can directly target DCs in vivo and be used to develop TAA-based vaccines. Another strategy involves ex vivo modification of DCs and their application for immunization. DCs isolated from bone marrow and transduced with SFV VLPs expressing cytokines or specific cDNAs from melanoma or glioma cell lines (B16 cDNA or 203 glioma cDNA vectors) provide protection from tumor challenge in brain tumor models [245]. Therapeutic vaccination of mice prolonged the overall survival of mice with established brain tumors. TAAs + Antibodies Synergistic therapeutic effects were obtained using a VEE/ TRP-2 vector in combination with either antagonist anti-CTLA-4 or agonist anti-GITR immunomodulatory mAbs [246]. The therapeutic combined treatment with VEE/TRP-2 VLPs and either anti-GITR or anti-CTLA-4 induced complete tumor regression in 90% and 50%, respectively, of mice bearing B16 mouse melanoma, compared to 20% treatment with the VEE/TRP-2 vaccine alone. Moreover, a second tumor challenge in surviving animals three months later did not produce any tumors, indicating the induction of a strong memory response. The agonist anti-GITR mAb promoted more efficient induction of humoral immunity against TRP-2 and stimulated the recruitment of phagocytes at the tumor site, whereas selective intratumoral accumulation of non-cytotoxic CD4+Foxp3 T cells expressing the negative co-stimulatory molecule (programmed

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cell death protein-1, PD-1) and common Treg-associated markers were observed in anti-CTLA-4 mAb-treated mice. VEGFR-2 + Cytokines The vascular endothelial growth factor receptor-2 (VEGFR-2) is a known therapeutic target because it is required for neovascularization within tumors and is important for tumor growth, invasion, and metastasis. VEGFR-2-expressing vectors induce humoral responses against VEGFR-2. Moreover, VEGF-2 receptors can also be expressed by cancer cells, e.g., breast and colon cancers [247, 248], suggesting that anti-VEGFR therapy may simultaneously target endothelial and tumor cells in those types of cancers. Five immunizations with SFV10-E VLPs expressing VEGFR-2 significantly inhibited both tumor growth and pulmonary metastatic spread in mice with pre-existing 4T1 tumors [249]. Similar results were obtained in a CT26 mouse colon cancer model. Microvessel density analysis demonstrated that immunization with SFVVEGFR-2 VLPs led to significant inhibition of tumor angiogenesis. Moreover, co-immunization of mice with SFV VLPs encoding VEGFR-2 and IL-4 as enhancers of the TH2 response led to the production of high titers of anti-VEGFR-2 antibodies and prolonged mice survival compared to co-immunization with VEGFR-2 and IL-12 or VEGFR-2 alone in both 4T1 and CT26 models. Although the combination of VEGFR-2 and IL-12 expression was inefficient [250], the SFV replicon-based DNA vector pSVKVEGFR2-GFc-IL12, which expresses VEGFR-2 and IL-12, was recently co-immunized with the SFV DNA vector expressing survivin and chorionic gonadotropin antigens in a B16 mouse melanoma model [250]. Combined DNA immunization simultaneously elicited efficient humoral and cellular immune responses against survivin, β-hCG and VEGFR2. This multimodal DNA vaccine inhibited tumor growth and improved the survival rate. Cytokines + Antibodies A promising strategy in cancer therapy is based on targeting specific pathways with immune checkpoint inhibitors. Immune checkpoints are mechanisms for preventing autoimmunity in peripheral tissue and regulating inflammatory responses [251]. Immune checkpoint inhibition with anti-CTLA-4 and anti-PD-1 antibodies has improved the survival of patients with metastatic melanoma, lung cancer and renal cancer [252]. The possible synergistic effects of these agents in combination with alphaviral vectors expressing cytokines were evaluated in several recent studies. Intratumoral injection of SFV/IL-12 VLPs synergized with systemic administration of an anti-PD-1 monoclonal antibody to induce tumor regression and prolong the survival of mice with MC38 mouse colon cancer and bilateral B16-OVA melanoma [253]. The results confirmed a synergistic therapeutic effect of SFV-IL12 with PD-1 blockade both against SFV/IL12 intratumorally injected tumors and distantly implanted lesions that were not injected with the viral SFV/IL12 vector in a bilateral model. Overexpression of the PD-L1 ligand on cancer cells was observed and was mediated by increased levels of IFN-γ in response to virus-driven IL-12 expression. Therefore, the synergistic effect was achieved by a well-timed targeted PD-L1 blockade on tumor cells and the induction of more potent antitumor cytotoxic T cell responses. Immunostimulatory monoclonal antibodies (mAbs) directed against CD137 to enforce T cell co-stimulation [254] were also synergistic with SFV-IL12 in poorly immunogenic B16 melanoma (B16-OVA and B16.F10) and TC-1 lung carcinoma models [255]. In this study, more than 70% of tumors were eradicated in animals receiving combined treatment with SFV-IL-12 (i.t.) and the agonist CD137 mAb (i.p.), in contrast to 7-37% tumor eradication in single treatments. Moreover, all animals in which tumors were eliminated remained tumor-free until the end of the study four months later. Remarkably, there was synergistic in vivo enhancement of CTLmediated immunity against the tumor antigens OVA and tyrosine-


related protein-2 (TRP-2), which led to long-lasting tumor-specific immunity against re-challenge. Taken together, these studies suggest that combination therapies that target the tumor mechanisms of immune evasion (e.g., removing the suppressive effect via PD1/PD-L1 neutralization) with activation of normal immune cell functionality (e.g., T cell activation) may provide optimal benefits for patients. Alphaviral Vectors + Anti-cancer Chemical Compounds Several studies have demonstrated a synergy between alphaviral vectors and chemotherapeutic agents. The efficacy of pSINCP/neu DNA and VEE/neu VLP vaccines was enhanced by pre-treating mice with doxorubicin and paclitaxel in a Her2/neuexpressing A2L2 breast cancer model [189]. Administration of doxorubicin prior to pSINCP/neu DNA and VEE/ neu VLPs vaccination led to a significant delay in tumor growth. Although doxorubicin has been established as a standard adjuvant therapy for breast cancer treatment, mice that received chemotherapy alone did not exhibit reductions of tumor growth. Interestingly, combination with paclitaxel enhanced the effectiveness of only the VEE/neu VLPbased vaccine. In another study, mice that were pre-treated with fluorouracil exhibited significantly increased intratumoral SFV-mediated transgene expression (Luc) in a 4T1 mouse breast cancer model mediated by enhancements in tumor vascular permeability and the inhibition of anti-viral type I IFN responses [256]. The combination of paclitaxel and SIN/LacZ VLP treatments in SCID mice bearing human ovary ES-2/Fluc tumors exhibited enhanced therapeutic effects for survival and inhibition of tumor growth [152]. The increase in blood vessel permeability induced by paclitaxel was postulated to enhance the intratumoral distribution and therapeutic effects of the SIN vectors. Reprogramming, or the functional inhibition of myeloidderived suppressor cells (MDSCs), represents another strategy in cancer treatment. The small-molecule inhibitor of tyrosine kinases sunitinib, which negatively regulates immunosuppressive MDSCs and Treg populations, was applied in combination with the previously described SFVeE6,7 vaccine in a mouse TC-1 cervical cancer model [257]. The combined treatment strongly enhanced the antitumor effect of the SFV vaccine, with a significant increase in E7specific CTLs and the depletion of MDSCs in the tumor environment. Moreover, triple treatment with sunitinib, SFVeE6,7 particles and single low-dose (14 Gy) tumor irradiation enhanced the immunotherapeutic antitumor effect, blocked tumor development and led to 100% survival of tumor-bearing mice [258]. Combining oncolytic SIN vector therapy with the topoisomerase inhibitor irinotecan (CPT-11) resulted in long-term survival of approximately 35% of SCID mice bearing ES2 human ovarian cancer [259]. Importantly, single-agent treatments did not result in long-term survival, and the combinatorial therapy was effective only in the presence of NK cells. Flow cytometry analysis, bioluminescent imaging and survival experiments demonstrated that SIN and CPT-11 utilize non-overlapping natural killer (NK)cell-dependent and -independent anti-cancer mechanisms. Bacteria-based Adjuvants + Alphaviral Vectors Bacterial infections of tumors induce in situ inflammation and trigger a versatile immune response that leads to tumor growth inhibition [260, 261]. Attenuated Salmonella typhimurium and genetically modified E. coli strains can elicit antitumor effects, particularly in combination with the expression of cytotoxic inducing factors or immunostimulatory cytokines [262, 263]. Bacteria-based neoadjuvant therapy for mouse metastatic breast cancer (4T1) was recently evaluated in combination with the SFV/IL-12 VLP vector [264]. SFV-IL-12 and the aroC - attenuated S. typhimurium strain LVR01 were intratumorally administered into 4T1 primary tumors, followed by surgical resection. A synergic antitumor effect of the combined therapy was observed compared to monotherapy, and


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metastasis-free and long-term survival was achieved in 90% of treated animals. Moreover, the therapeutic effect was observed only when SFV/IL-12 was administered before LVR01, whereas administration of LVR01 before SFV/IL-12 resulted in insignificant antitumor activity. Regardless, this is a promising therapeutic strategy for preventing and/or eradicating pre-operatory metastasis. 4. BIO-DISTRIBUTION AND TUMOR TARGETING Alphaviruses have a broad host range, which raises concerns about the use of recombinant particles for cancer therapy. For intratumoral injection, the risk of spreading replication-deficient particles is negligible. Moreover, the opposite problem, poor virus dissemination, remains to be resolved. Viruses are relatively large, mostly charged particles with a diameter of 50-200 nanometers. These properties, combined with characteristic denser stroma in solid tumors, result in impaired mobility in tumor tissue and, consequently, poor spread and low infectivity [265]. A prospective approach for improving tumor transduction efficiency was demonstrated using chimeric adenovirus-alphavirus vector expressing relaxin, a peptide hormone that decreases the synthesis and secretion of interstitial collagens and increases the expression of matrix metalloproteinases in tumor xenografts [266]. Another interesting approach for increasing viral spread in tumors was demonstrated by expressing a virus envelope-derived fusogenic membrane glycoprotein with an engineered SIN virus vector, which induced cell membrane fusion between the infected and uninfected glioma cells and subsequently resulted in syncytia formation, leading to efficient viral spread within the tumor [267]. However, for systemic delivery and, specifically, the use of oncolytic replication-proficient vectors carrying anticancer or toxic genes, there is a considerable risk of damage to normal tissue. Consequently, SIN particles have been subjected to tumor targeting by engineering IgG-binding domains into the E2 envelope protein [268]. These chimeric SIN particles improved the transduction of host cells treated with a monoclonal antibody against surface proteins, whereas a 105-fold reduction in BHK cell transduction was observed. A subsequent study demonstrated that SIN particles exhibited natural tumor targeting after systemic administration in mice implanted with human xenografts [151]. Intraperitoneal injections of SIN particles expressing the luciferase reporter gene showed targeted tumor expression in fibrosarcomas in the tails of injected mice. Different proteins have been proposed as receptor candidates for alphavirus infection [269, 270]. However, because alphaviruses infect genetically divergent cells, they may utilize multiple proteins as receptors or alternative entry pathways in different cells. Furthermore, SIN particle tumor tropism in human xenograft models was not defined by SIN receptor levels but by the IFN response in tumors because cells with defects in either IFN production or signaling exhibited strong susceptibility to SIN particles [230]. Surprisingly, no tumor targeting was observed for SFV particles after systemic delivery [271]. However, despite the broad distribution, preferential intratumoral expression of the SFV vector upon systemic injections was achieved with an optimized reduced virus dose [272]. To increase tumor targeting, another approach involved engineering liposome encapsulated nucleic acids and viral particles [273]. Systemic administration of liposome-encapsulated SFVLacZ particles resulted in enhanced β-galactosidase expression in tumor tissues. Because alphavirus vectors are RNA viruses, selective replication in tumor cells cannot be designed by the standard use of tissue/tumor-specific RNA polymerase II-dependent promoter sequences, except for DNA layered vectors. An alternative option is to ensure replication selectivity at the translational level using internal ribosome entry sites (IRES) to regulate transgene expression. Interestingly, in cells where translation is regulated by IRES sequences, many sites are located in genes encoding proteins respon-


sible for regulating cell proliferation and survival/apoptosis and whose misregulation is frequently associated with cancer [274]. The mechanisms of IRES-guided translation are employed by some RNA viruses, such as hepatitis C, polio, encephalomyocarditis, rhinovirus, and picornavirus. There have been successful attempts to use IRES sequences derived from other positive-polarity RNA viruses to differentially regulate the expression of viral structural proteins of alphaviruses [275, 276]. 5. BIOSAFETY ASPECTS As described in the introduction (section 1. Biology of alphaviruses) alphaviruses have been linked to disease in both domestic animals and humans [7-9]. In this context, EEE, WEE and VEE can cause encephalitis [6, 9]. SFV and SIN have been associated with fever epidemics [11, 12]. Moreover, CHIK has been linked to polarthritis epidemics [16, 17]. Keeping these potential hazards in mind, engineering of alphavirus vectors has been based on attenuated or avirulent laboratory strains [13]. Even so, for any in vivo application of viral vectors, a biosafety risk assessment is necessary. The use of conventional alphavirus expression and helper vectors has been associated with the homologous recombination and generation of a small fraction of replication-proficient particles [9]. To address this shortcoming, the second-generation SFVHelper2 vector was engineered. This vector renders the generated particles conditionally infectious due to the presence of three point mutations at the cleavage sites of E2 and E3 proteins in the p62 precursor [277]. Infectious particles are obtained only after αchymotrypsin treatment, which substantially reduces the rate of homologous recombination and production of replication-proficient particles. To reduce recombination events to negligible levels, split helper systems in which the capsid and envelope genes are placed on separate helper vectors have been engineered for SFV [278] and SIN [279]. Furthermore, to increase the safety and the controlled propagation of the replication-competent SIN vector, a suicide gene encoding a widely used herpes simplex virus type-1 thymidine kinase (TK) was incorporated into the viral genome by fusion with the nsP3 subunit of the viral replicase [280]. Administering the prodrug ganciclovir (GCV) shut off virus propagation in mice that had intraperitoneally received the oncolytic SIN-TK. Therefore, incorporating an inducible suicide gene provides an additional layer of protection for achieving controlled propagation of the vector in vivo. Incorporating tissue-specific miRNAs into vectors can alter the tissue specificity of virus replication. This approach can be used to improve the safety of oncolytic viruses by restricting their replication in unwanted tissues, such as the liver. To minimize the neurotoxicity of the SFV4 vector, six tandem targets for the neuronspecific miR124 were inserted between the viral nonstructural protein 3 and 4 (nsP3 and nsP4) genes [281]. Intraperitoneally administered SFV4-miRT124 particles exhibited attenuated vector spread within the central nervous system and greatly increased the survival of BALB/c mice. Moreover, intracranial infections in adult mice with SFV4-miRT124 revealed greatly reduced infection of neurons but infection of oligodendrocytes in the corpus callosum. Incorporating multiple miRNA targets might be a feasible and promising strategy for generating safe and targeted oncolytic alphavirus virotherapy agents. 6. CLINICAL TRIALS Although a few immunization studies have been carried out in animal models only a limited number of clinical trials have been conducted for alphaviruses. In an immunotherapy phase I trial, liposome encapsulated SFV particles expressing interleukin-12 (IL12) were intravenously administered to melanoma and kidney carcinoma patients [273]. Transient 5-fold increase in plasma levels of IL-12 was observed for 4-7 days after administration at the maxi-

Zajakina et al.

mum tolerated dose (MTD) of 3 x 109 encapsulated particles per m2. Higher SFV-IL12 doses generated fever responses in patients. The study showed no liposome- or SFV-related toxicity. Repeated systemic administration was possible as liposome-encapsulation prevented SFV particles from being recognized by the host immune system. Overall, the safety profile of administered encapsulated SFV particles was good. In another approach, a Phase I randomized, double-blind clinical trial for an alphavirus-based two-component vaccine expressing cytomegalovirus (CMV) gB or pp65/1E1 fusion protein was administered intramuscularly or subcutaneously in CMV seronegative adult volunteers [282]. Immunization elicited neutralizing antibody and multifunctional T cell responses against CMV antigens. No clinically important changes occurred and only mild to moderate local reactions were observed after vaccination. Moreover, patients with metastatic cancer were subjected to repeated administration of alphavirus particles expressing the CEA in another clinical trial [283]. The elicited CEA-specific antibodies were able to mediate antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. Moreover, the overall survival was extended in patients with CEA-specific antibodies. In another clinical trial, VEE replicons expressing PSMA were administered to patients with metastatic cancers at five doses of 0.9 x 107 IU or 3.6 x 107 IU [215]. No PSMA-specific cellular response was obtained at the lower dose and only a weak PSMA-specific signal was detected by ELISA. More disappointingly, the higher dose showed no PMSA-specific response. Although neither clinical benefit nor robust immune responses were obtained in the study, the immunization with VEE-PMSA particles was well tolerated. The lack of strong immune responses in the study might be related to the dosing and still requires some optimization. CONCLUSION AND FUTURE PROSPECTS Immunotherapy has become an important approach for modern cancer therapy from both prophylactic and therapeutic perspectives. As with all forms of therapeutic interventions, delivery has been a major challenge. Applying viral vectors for immunogens has proven favorable by ensuring intratumoral delivery and reduced toxicity compared with direct administration of recombinant proteins. In this context, alphavirus vectors are attractive because they provide high levels of transient gene expression, which is considered ideal for immunization. The lack of existing antibodies in humans and the low immunogenicity of the vectors are also important features. The delivery of alphavirus particles, RNA replicons and layered DNA vectors elicits strong immune responses against TAAs, cytokines, growth factors and a number of antibodies. Furthermore, combination therapy using these reagents and co-administration with chemotherapeutics and bacteria-based adjuvants has increased the range of potential applications. Additionally, the expression of molecules that modulate the tumor stroma environment, decrease the density of connective tissue, or, alternatively, permit the dissemination of replication-deficient virus vectors through the specific fusion of infected tumor cells to adjacent non-infected cells are interesting approaches for future studies to significantly enhance the efficacy of virus therapy. Taking into account recent advances in understanding the mechanisms of immune escape and the tumor supporting microenvironment, new improved multistep immunotherapeutic strategies have to be designed. Obviously, careful host immune profiling in terms of alphavirus infection is required with sequential control of the success of therapeutic intervention in each step in order to control the tumor suppressive environment, as well as to present efficiently tumor-specific antigens and subsequently to recruit specific CTLs to tumors. The progress in more than 25 years of research on alphaviruses has allowed the design of extremely efficient tumor targeting vectors to break tumor immune-tolerance and reprogram


the tumor microenvironment to be used in combination with such drugs, as immune checkpoint and growth factor/receptor inhibitors. The potential use of IRES elements for guided selective alphavirus RNA translation together with tissue-specific miRNAs and expression of specific chemokines, which are capable for instance of inducing polarization of tumor-associated macrophages towards the M1 phenotype, are prospective strategies still needed to be realized. The advances in vector design allowing specific targeting and selective tissue replication together with employment of new more “aggressive” oncolytic viral vectors would definitely lead to more efficient approaches in cancer treatment. Immunization studies in rodent models have already resulted in protection against challenges with tumors and encouraged researchers to conduct clinical trials in cancer patients. Although more studies utilizing innovative approaches are required, alphavirus-based immunotherapy holds a great promise in the battle against cancer. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS This work was supported by Norway Grants 2009-2014 under project contract NFI/R/2014/051.

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