Single-stranded RNA viruses such as alphaviruses, flaviviruses, measles viruses and rhabdoviruses are characterized by their capacity of highly efficient self-amplification of RNA in host cells, which make them attractive vehicles for vaccine development. Particularly, alphaviruses and flaviviruses can be administered as recombinant particles, layered DNA/RNA plasmid vectors carrying the RNA replicon and even RNA replicon molecules. Self-amplifying RNA viral vectors have been used for high level expression of viral and tumor antigens, which in immunization studies have elicited strong cellular and humoral immune responses in animal models. Vaccination has provided protection against challenges with lethal doses of viral pathogens and tumor cells. Moreover, clinical trials have demonstrated safe application of RNA viral vectors and even promising results in rhabdovirus-based phase III trials on an Ebola virus vaccine. Preclinical and clinical applications of self-amplifying RNA viral vectors have proven efficient for vaccine development and due to the presence of RNA replicons, amplification of RNA in host cells will generate superior immune responses with significantly reduced amounts of RNA delivered. The need for novel and efficient vaccines has become even more evident due to the global COVID-19 pandemic, which has further highlighted the urgency in challenging emerging diseases.

Self-Amplifying RNA Virus-Based Expression

Although efficient expression systems have been developed for alphaviruses, flaviviruses, MVs and rhabdoviruses, the differences in the polarity of the ssRNA genome have required alternative engineering. In the case of alphaviruses and flaviviruses, the procedure is straightforward, whereas MVs and rhabdoviruses require the utilization of packaging cell lines and reverse genetics. The Semliki Forest virus (SFV)-based expression system is one of the most commonly applied alphavirus expression systems.

Fig. 1 Semliki Forest virus-based expression systems.

Alphaviruses belong to the family of Togaviruses. Particularly, SFV, Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE) have been used for the engineering of expression vector systems. In this context, engineering of an expression vector carrying the SFV nonstructural protein genes (nsP1-4), where the gene of interest can be inserted downstream of the strong 26S subgenomic promoter allows in vitro transcribed RNA to be directly translated in cell lines and in vivo. Alternatively, replacement of the SP6 or T7 RNA polymerase promoter upstream of the nsP1-4 region with a cytomegalovirus (CMV) promoter permits direct use of plasmid DNA in vitro or in vivo. Another possibility is to co-transfect in vitro transcribed RNA from the expression vector and a helper vector expressing the SFV structural protein genes into mammalian host cells for production of replication deficient SFV particles. The reason for replication deficiency relates to the presence of the packaging signal in the nsP2 region of the expression vector, which prevents the packaging of the RNA from the helper vector. Additionally, to reduce the formation of any replication proficient SFV particles, the second generation pSFV-Helper 2 vector was engineered. Furthermore, for total elimination of RNA recombination and generation of replication proficient SFV particles a two-helper system with the capsid and spike protein genes on separated vectors was applied. Finally, a full-length SFV genome with a second 26S subgenomic promoter and gene of interest introduced either downstream of the nsP genes or the structural genes can be applied, keeping in mind that replication proficient particles will be generated, obviously enhancing biosafety risks. In any case, the options and flexibility are excellent as alphavirus vectors can be used for vaccine development in the form of naked RNA replicons, DNA plasmid vectors and recombinant viral particles.

Vaccines Against Infectious Diseases

There are numerous examples of preclinical immunization studies against infectious diseases conducted with self-replicating RNA virus vectors. In the context of Dengue virus (DENV), a single immunization with a tetravalent vaccine based on VEE particles expressing the ectodomain of the DENV E protein (E85) elicited neutralizing antibodies and T cell responses for each of four serotypes tested.

Filoviruses have for obvious reasons received a lot of attention due to such members as Ebola virus (EBOV), presenting urgent needs for the development of novel vaccines especially during the Ebola virus disease (EVD) outbreak in 2014-2016. In this context, KUN virus-like particles (VLPs) expressing the EBOV glycoprotein with the D637L mutation (GP/D637L), which by displaying a cleavage site improved cleavability and shedding of GP, were subcutaneously injected into nonhuman primates.

In the context of arenaviruses, development of Lassa virus (LASV) vaccines has been initiated by expressing LASV glycoproteins from VSV vectors. Immunization studies in guinea pigs and macaques provided protection against LASV originating from Liberia, Mali and Nigeria. In another study, the wild-type LASV glycoprotein (GPCwt) and a non-cleavable C-terminally deleted modification (ΔGPfib) expressed from individual VEE 26S subgenomic promoters were immunogenic and protective in immunized mice.

Lentiviruses have also been commonly chosen as vaccine targets, obviously because of human immunodeficiency virus (HIV) causing AIDS. For instance, recombinant SFV particles expressing HIV-1 envelope glycoprotein (Env) were applied for immunization studies in mice in comparison to a DNA vaccine and recombinant Env gp160 protein. The highest antibody titers were observed in animals immunized with SFV particles. Furthermore, an SFV RNA replicon vector carrying the HIV-1 Env gene was applied for intramuscular injection into mice. The immunization resulted in induction of Env-specific antibody responses in four out of five mice. Moreover, the monoclonal antibody 12H2 directed against gp41 was produced.

Due to the recurrent annual threats of epidemics, influenza viruses have been sought after targets for vaccine development. In this context, intravascular administration of SFV particles expressing the influenza virus nucleoprotein (NP) resulted in systemic immune responses in mice. Furthermore, intranasal delivery elicited mucosal immune responses.

Vaccines Against Cancer

Cancer therapy and immunotherapy are areas of great potential for self-amplifying RNA virus vectors. Not surprisingly, most types of cancers have been targeted in preclinical settings. In the case of brain tumors, a retargeted MV vector with CD46 and signaling lymphocyte activation molecule (SLAM) ablating mutations in the HA protein combined with a single-chain antibody against the epidermal growth factor receptor (EGFR) showed potent antitumor activity against EGFR- or EGFRvIII-overexpressing primary glioblastoma multiforme (GBM) cell lines. Intratumoral administration in orthotopic GBM12 xenografts resulted in tumor regression and significantly prolonged survival. Alphavirus vectors have also been applied for cancer therapy. In this context, SFV vectors expressing endostatin in comparison to SFV-LacZ and retrovirus-based endostatin expression provided superior inhibition of tumor growth and reduction in intratumoral vascularization in mice with B16 brain tumor xenografts. Moreover, dendritic cells (DCs) transduced with SFV particles expressing interleukin-18 (IL-18) were administered intratumorally into mice bearing B16 brain tumors in combination with systemic administration of IL-12. The SFV-based treatment resulted in enhanced induction of T helper type 1 responses from tumor specific CD4 and CD8 T cells and natural killers and antitumor immunity. In another study, neuron-targeting micro-RNA sequences (miRT124) introduced into the SFV4 strain generated increased oncolytic potency in human glioblastoma cell lines and resulted in virus replication in tumors, significant inhibition of tumor growth and prolonged survival after intraperitoneal administration in C57BL/6 mice implanted with CT-2A orthotopic gliomas. A chimeric vector was engineered, where the VSV G protein was replaced by the CHIKV envelope proteins (E3-E2-6K-E1). The VSVΔG-CHIKV vector selectively infected and eliminated tumors and the survival of tumor-bearing mice was extended from 40 to 100 days. Moreover, the chimeric virus also targeted intracranial xenografts from melanoma patients and intravenous administration only infected tumor cells and selectively infected mouse melanoma cells within the brain.

Clinical Trials

In the context of clinical trials, the number of conducted studies using self-amplifying RNA virus vectors are relatively modest compared to for instance adenovirus and retrovirus vectors. In a randomized, double-blind phase I trial, 40 CMV seronegative healthy volunteers were intramuscularly or subcutaneously injected with VEE particles expressing the CMV glycoprotein B or the fusion between CMV pp65 and IE1 proteins. The vaccinations with a lower dose of 1 × 10⁷ infectious units (IU) and a higher dose of 1 × 10⁸ IU were well tolerated with only mild to moderate local reactogenicity, minimal systemic reactogenicity and no clinically important changes in laboratory parameters. Direct IFN-γ responses to CMV antigens were obtained in all vaccinated subjects. The procedure was safe and neutralizing antibodies and multifunctional T cell responses were generated against all three CMV antigens important for protective immunity. In another randomized, double-blind, placebo-controlled phase I trial, HIV-negative volunteers in the US and South Africa were subjected to subcutaneous injection of escalating doses of VEE particles expressing the non-myristoylated form of the HIV-1 subtype C Gag protein. The vaccine was well tolerated with only modest local reactogenicity. However, five serious adverse events were reported, albeit none of them were considered to be related to the vaccine. Disappointingly, the immune responses comprised only low levels of binding antibodies and T cell responses at the highest dose of 1 × 10⁸ IU.

In conclusion, self-amplifying RNA virus vectors provide the flexibility of utilizing RNA replicons, recombinant VLPs or DNA replicon plasmids for immunization studies leading to robust antibody responses and protection against challenges with lethal doses of pathogens or tumor cells. Although so far, clinical responses have been relatively modest, vector development, dosage optimization and delivery improvement will support the production of efficient vaccines in the near future. These needs are even more crucial today with the current COVID-19 pandemic.