During the last decade, immunotherapy has become an attractive alternative for cancer therapy [1]. In this context, viral vectors have also proven useful for immunotherapeutic applications [2]. Alphaviruses have frequently been engineered for the overexpression of suitable antigens and immunostimulatory genes for vaccine development and cancer therapy [3]. Additionally, the expression of cytotoxic and antitumor genes has been used for cancer therapy applications. Semliki Forest virus (SFV) [4], Sindbis virus (SIN) [5] and Venezuelan equine encephalitis virus (VEE) [6] are most commonly used for the engineering of expression systems. Additionally, the naturally occurring oncolytic alphavirus M1 [7] and engineered oncolytic versions based on SFV and SIN vectors [8] have been utilized for cancer therapy. The evaluation of efficacy in appropriate animal models has provided proof of concept before conducting clinical trials.

2. Alphavirus Lifecycle and Expression Vector Systems

Alphaviruses possess an enveloped structure of capsid and spike proteins encapsulating a single-stranded RNA (ssRNA) genome of positive polarity [9]. Upon the infection of host cells, the alphavirus ssRNA is released into the cytoplasm, where translation can immediately occur requiring no delivery of RNA to the nucleus as is the case for other RNA viruses such as the influenza virus and DNA viruses (Figure 1). In the cytoplasm, efficient self-replication occurs through a minus-strand RNA template leading to the accumulation of approximately 10⁶ copies of subgenomic RNA per cell. Together with the utilization of the highly efficient 26S subgenomic promoter, high-level expression of viral proteins occurs [10]. The RNA self-replication and high-level expression of alphavirus structural proteins generate high-titer virus progeny. Nucleocapsids comprising the capsid protein harboring full-length alphavirus RNA are transported to the cell surface, where the envelope proteins are attached, and mature viral particles are released by budding. In the case of expression systems, the focus is on the expression of heterologous genes (Figure 2). In the context of replication-deficient alphavirus particles, the structural protein genes have been replaced by the gene of interest (GoI), and a helper vector is engaged in providing the structural proteins in trans (Figure 2A). Co-transfection of in vitro transcribed RNA from expression and helper vectors into baby hamster kidney (BHK) cells leads to the production of recombinant particles. As the RNA packing signal is located in one of the genes coding for the non-structural proteins (nsPs), in the nsP2 gene of SFV and nsP1 of SIN [11], uniquely RNA from the expression vector is packaged into viral particles, providing expression of the GoI but not the structural protein genes and thereby, eliminating any production of viral progeny. In contrast, introduction of a second 26S subgenomic promoter and the GoI into the full-length alphavirus RNA genome, either downstream of the nsP or the structural protein genes, generates replication-competent particles capable of both high-level GoI expression and viral progeny production (Figure 2B). In addition to the application of recombinant particles, RNA replicons can also be used for GoI expression. As has been demonstrated for the recent BNT162b2 [12] and mRNA-1273 [13] COVID-19 vaccines, RNA-based delivery is highly efficient. However, in contrast to this conventional mRNA approach, alphavirus RNA replicons provide the additional advantage of RNA self-amplification leading to superior expression levels. Moreover, replacement of the SP6 RNA polymerase promoter by a CMV promoter, DNA replicon vectors for GoI expression (Figure 2C) have been engineered for the transfection of cell lines and in vivo administration [14]. The use of DNA replicons eliminates any risk of the production of new virus particles but relies on the less efficient delivery of DNA compared to viral vectors. Moreover, DNA molecules must be delivered to the nucleus for the in vivo transcription of RNA (Figure 2C).

3. Alphavirus-Based Immunotherapy for Cancer

In the context of cancers, alphaviruses have been frequently used for prophylactic and therapeutic applications. Immunization with alphavirus vectors overexpressing tumor-associated antigens (TAAs) has been a common approach for cancer vaccine development. This approach has been used to provide both prevention against tumor challenges and tumor regression and eradication. Moreover, overexpression of cytotoxic and antitumor genes has been evaluated for cancer therapy. The delivery of immunostimulatory genes from alphavirus vectors has served the means of cancer immunotherapy. Moreover, alphaviruses induce apoptosis through activation of caspases in infected cells [15], which has resulted in tumor regression after administration of alphaviruses carrying no therapeutic genes and has allowed the use of vectors with reporter genes to verify and localize expression in animal tumor models. Finally, engineered or naturally occurring oncolytic alphaviruses have demonstrated tumor cell-specific killing in animal models [16]. Examples of cancer vaccinations, cancer therapy and immunotherapy are given below and summarized in Table 1.