Semliki Forest Virus Particles Research Articles

Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus

Semliki Forest virus (SFV) is a potential cancer gene therapy vector capable of providing high and transient expression of heterologous proteins in mammalian cells. However, SFV has shown suboptimal transduction levels in several cancer cell types as well as wide biodistribution of SFV has been observed after in vivo applications. Magnetic nanoparticles (MNPs) have been shown to increase cell transduction with several viral vectors in vitro under an external magnetic field and enhance magnetically guided viral vector delivery.Here, we examined a panel of MNPs for enhanced cancer cell transduction with SFV vector. Magneto-transduction using positively charged MNPs increased Semliki Forest virus transduction in TS/A mouse mammary carcinoma cells in vitro in the presence of fetal bovine serum. Positively charged MNPs efficiently captured SFV particles independently of capturing medium, and MNPs-SFV complexes were successfully separated from suspension by magnetic precipitation. These results reveal the potential application of MNPs for enhanced gene delivery by SFV vector as well as proposes magnetic precipitation for efficient concentration of SFV particles from different media.

Role of TSPAN9 in Alphavirus Entry and Early Endosomes.

Alphaviruses are small enveloped RNA viruses that infect cells via clathrin-mediated endocytosis and low-pH-triggered fusion in the early endosome. Using a small interfering RNA (siRNA) screen in human cells, we previously identified TSPAN9 as a host factor that promotes infection by the alphaviruses Sindbis virus (SINV), Semliki Forest virus (SFV), and chikungunya virus (CHIKV). Depletion of TSPAN9 specifically decreases SFV membrane fusion in endosomes. TSPAN9 is a member of the tetraspanin family of multipass membrane proteins, but its cellular function is currently unknown. Here we used U-2 OS cells stably overexpressing TSPAN9 to show that TSPAN9 is localized at the plasma membrane and in early and late endosomes. Internalized SFV particles colocalized with TSPAN9 in vesicles early during infection. Depletion of TSPAN9 led to reductions in the amounts of the late endosomal proteins LAMP1 and CD63 and an increase in the amount of LAMP2. However, TSPAN9 depletion did not alter the delivery of SFV to early endosomes or change their pH or protease activity. Comparative studies showed that TSPAN9 depletion strongly inhibited infection by several viruses that fuse in early endosomes (SFV, SINV, CHIKV, and vesicular stomatitis virus [VSV]), while viruses that fuse in the late endosome (recombinant VSV-Lassa and VSV-Junin), including an SFV point mutant with a lower pH threshold for fusion (SFV E2 T12I), were relatively resistant. Our data suggest that TSPAN9 modulates the early endosome compartment to make it more permissive for membrane fusion of early-penetrating viruses. Alphaviruses are spread by mosquitoes and can cause serious human diseases such as arthritis and encephalitis. Recent outbreaks of CHIKV infection are responsible for millions of cases of acute illness and long-term complications. There are no vaccines or antiviral treatments for these important human pathogens. Alphaviruses infect host cells by utilizing the endocytic machinery of the cell and fusing their membrane with that of the endosome. Although the mechanism of virus-membrane fusion is well studied, we still know relatively little about the host cell proteins that are involved in alphavirus entry. Here we characterized the role of the host membrane protein TSPAN9 in alphavirus infection. TSPAN9 was localized to early endosomes containing internalized alphavirus, and depletion of TSPAN9 inhibited virus fusion with the early endosome membrane. In contrast, infection of viruses that enter through the late endosome was relatively resistant to TSPAN9 depletion, suggesting an important role for TSPAN9 in the early endosome.

BST2/tetherin inhibition of alphavirus exit.

Alphaviruses such as chikungunya virus (CHIKV) and Semliki Forest virus (SFV) are small enveloped RNA viruses that bud from the plasma membrane. Tetherin/BST2 is an interferon-induced host membrane protein that inhibits the release of many enveloped viruses via direct tethering of budded particles to the cell surface. Alphaviruses have highly organized structures and exclude host membrane proteins from the site of budding, suggesting that their release might be insensitive to tetherin inhibition. Here, we demonstrated that exogenously-expressed tetherin efficiently inhibited the release of SFV and CHIKV particles from host cells without affecting virus entry and infection. Alphavirus release was also inhibited by the endogenous levels of tetherin in HeLa cells. While rubella virus (RuV) and dengue virus (DENV) have structural similarities to alphaviruses, tetherin inhibited the release of RuV but not DENV. We found that two recently identified tetherin isoforms differing in length at the N-terminus exhibited distinct capabilities in restricting alphavirus release. SFV exit was efficiently inhibited by the long isoform but not the short isoform of tetherin, while both isoforms inhibited vesicular stomatitis virus exit. Thus, in spite of the organized structure of the virus particle, tetherin specifically blocks alphavirus release and shows an interesting isoform requirement.

Semliki forest virus-based expression of recombinant GPCRs.

Due to their importance as targets for drug development, rapid and consistent high-level production of G protein-coupled receptors (GPCRs) has become an essential part of drug discovery. Alphaviruses, particularly recombinant Semliki Forest virus (SFV) particles, have provided the means for expression of a number of GPCRs in a broad range of mammalian host cell lines for pharmacological characterization by determination of receptor binding activity and functional coupling to G proteins. The rapid high-titer virus particle production has made it possible to study a large number of GPCRs in parallel. Moreover, large-scale production in adherent and suspension cultures of mammalian cells has provided sufficient amounts of GPCRs for purification and subsequent structural studies. Furthermore, the high preference for neuronal delivery of SFV particles has allowed functional and localization studies of recombinant proteins in hippocampal slice cultures, in primary neurons, and in vivo.

A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

Semliki Forest virus biodistribution in tumor-free and 4T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon

Semliki Forest virus (SFV) vectors are promising tools for cancer gene therapy because they ensure a high level of transgene expression and a rapid and strong cytopathic effect. However, broad tissue tropism and transient expression make it more difficult to develop an optimal cancer treatment strategy. In this study, we have compared the distribution of recombinant SFV particles (recSFV) and naked viral RNA replicon (recRNA) in tumor-free and 4T1 mammary tumor-bearing mice as a consequence of different vector administration strategies. The high potential of SFV recRNA as a biosafe approach for the development of therapeutic treatment was demonstrated. Intravenous (i.v.) inoculation of recRNA provided primary brain targeting in both tumor-free and 4T1 tumor mouse models, but local intratumoral inoculation revealed a high expression level in tumors. Moreover, we observed the predominant tumor targeting of recSFV at a reduced viral dose on i.v. and intraperitoneal (i.p.) virus inoculation, whereas the dose increase led to a broad virus distribution in mice. To prolong transgene expression, we have tested several i.v. and i.p. reinoculation strategies. A detailed evaluation of vector distribution and readministration properties could have an impact on cancer gene therapy clinical trial safety and efficacy.

Alphaviruses in Gene Therapy

Alphaviruses are enveloped single stranded RNA viruses, which as gene therapy vectors provide high-level transient gene expression. Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan Equine Encephalitis (VEE) virus have been engineered as efficient replication-deficient and -competent expression vectors. Alphavirus vectors have frequently been used as vehicles for tumor vaccine generation. Moreover, SFV and SIN vectors have been applied for intratumoral injections in animals implanted with tumor xenografts. SIN vectors have demonstrated natural tumor targeting, which might permit systemic vector administration. Another approach for systemic delivery of SFV has been to encapsulate replication-deficient viral particles in liposomes, which can provide passive targeting to tumors and allow repeated administration without host immune responses. This approach has demonstrated safe delivery of encapsulated SFV particles to melanoma and kidney carcinoma patients in a phase I trial. Finally, the prominent neurotropism of alphaviruses make them attractive for the treatment of CNS-related diseases.

Characterization of specific immune responses in cattle immunized with plasmid DNA construct and recombinant Semliki Forest virus particles encoding Cu,Zn superoxide dismutase (SOD) of Brucella abortus

Characterization of specific immune responses in cattle immunized with plasmid DNA construct and recombinant Semliki Forest virus particles encoding Cu,Zn superoxide dismutase (SOD) of Brucella abortus

Vaccination with recombinant Semliki Forest virus particles expressing translation initiation factor 3 of Brucella abortus induces protective immunity in BALB/c mice

Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 ( P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.

Evaluation of Brucella abortus DNA and RNA vaccines expressing Cu–Zn superoxide dismutase (SOD) gene in cattle

This study was conducted to evaluate the immunogenicity of a DNA or RNA vaccines encoding Brucella abortus Cu–Zn superoxide dismutase (SOD) in cattle. Intramuscular injection of plasmid DNA carrying Brucella SOD gene (pcDNA-SOD) into animals elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD IgG antibody with predominance of immunoglobulin G1 (IgG1) isotype over IgG2. In addition, the DNA vaccine elicited a specific T-cell-proliferative response. Furthermore, intraperitoneal injection of cattle with recombinant Semliki Forest virus particles carrying recombinant RNA encoding SOD (SFV-SOD) did not lead to the induction of SOD IgG 1 or 2 antibody, but induced specific T-cell activation. Both vaccines were able to induce a non-significant secretion of gamma interferon and did not induce the secretion of IL-4 or tumor necrosis factor (TNF)-α. These results suggest that SOD gene in a genetic vaccine formulation (DNA or RNA) might be of potential us as a vaccine to induce cell-mediated immunity in cattle. To our knowledge, this is the first study to evaluate a genetic vaccine against Brucella in cattle.

Semliki Forest virus vectors with mutations in the nonstructural protein 2 gene permit extended superinfection of neuronal and non-neuronal cells

Semliki Forest virus (SFV) vectors are widely used in neurobiological studies because they efficiently infect neurons. As with any viral vector, they possess a limited cloning capacity, so infection with different SFV vectors may be required to introduce multiple transgenes into individual cells. However, this approach is limited by superinfection exclusion. The authors examined marker expression in baby hamster kidney cells, mouse cortical neurons, and rat hippocampal neurons using different fluorophore-encoding vectors that are based on the wild-type SFV4 strain and on the less cytopathic SFV4(PD) mutant, which carries two point mutations in nonstructural protein 2. For every fluorophore tested, SFV4(PD) gave higher (up to 22-fold) expression compared to SFV4. In infections using two and three different vectors, SFV4 caused relatively few multifluorescent baby hamster kidney cells when applied at 0-s, 15-min, or 2-h intervals. In contrast, SFV4(PD) permitted significantly enhanced marker coexpression, resulting in 46% doubly and 21% triply fluorescent baby hamster kidney cells, and 67% to 8% doubly fluorescent cortical and hippocampal neurons. At 15-min or 2-h addition intervals, SFV4(PD) still permitted 23% to 36% doubly fluorescent baby hamster kidney cells. The increased efficiency of SFV4(PD) in coexpressing separate markers from different viral particles suggests that mutations in nonstructural protein 2 affect alphaviral superinfection exclusion. The results demonstrate that SFV4(PD) is well-suited to coexpress multiple proteins in neuronal and non-neuronal cells. This capability is particularly valuable to express the various components of heteromeric protein complexes, especially when the individual cDNAs cannot be combined into single SFV particles.

Inhibition of angiogenesis by a Semliki Forest virus vector expressing VEGFR-2 reduces tumour growth and metastasis in mice

Inhibition of tumour angiogenesis has been shown to restrict primary tumour growth and metastatic spread. This study examines the active induction of immune responses against tumour endothelial cells following immunization with recombinant Semliki Forest virus (rSFV) particles encoding murine vascular endothelial growth factor receptor-2 (VEGFR-2). This approach was tested in two murine tumour models, CT26 colon carcinoma and 4T1 metastasizing mammary carcinoma. Tumour growth and metastatic spread were shown to be significantly inhibited in mice that were prophylactically vaccinated or therapeutically treated with rSFV particles coding for VEGFR-2. Microvessel density analysis showed that immunization with rSFV led to significant inhibition of tumour angiogenesis. Therapeutic efficacy was found to be associated with the induction of an antibody response against VEGFR-2. Co-immunization of mice with rSFV particles encoding VEGFR-2 and interleukin (IL)-12 completely abrogated both the antibody response and the antitumour effect. However, co-immunization of mice with VEGFR-2 and IL-4 encoding particles was shown both to induce higher titres of anti-VEGFR-2 antibodies and lead to enhanced survival following tumour challenge when compared to mice vaccinated with VEGFR-2 particles alone. These findings indicate that active immunization with rSFV particles coding for VEGFR-2 can break immunological tolerance and could potentially be used as part of a novel treatment for cancer.

Involvement of Protein Synthesis and Degradation in Long-Term Potentiation of Schaffer Collateral CA1 Synapses

Expression of synaptic plasticity involves the translation of mRNA into protein and, probably, active protein degradation via the proteasome pathway. Here, we report on the rapid activation of synthesis and degradation of a probe protein with the induction of long-term potentiation (LTP) in the hippocampal Schaffer collateral CA1 pathway. The proteasome inhibitor MG132 significantly reduced the field EPSP slope potentiation and LTP maintenance without acutely affecting basal synaptic transmission. To visualize protein dynamics, CA1 pyramidal cells of hippocampal slices were transfected with Semliki Forest virus particles expressing a recombinant RNA. This RNA contained the coding sequence for a degradable green fluorescence protein with a nuclear localization signal (NLS-d1EGFP) followed by a 3'- untranslated region dendritic targeting sequence. NLS-d1EGFP fluorescence remained stable in the low-frequency test stimulation but increased with LTP induction in the cell body and in most dendritic compartments of CA1 neurons. Applying anisomycin, a protein synthesis inhibitor, caused NLS-d1EGFP levels to decline; a proteasome inhibitor MG132 reversed this effect. In the presence of anisomycin, LTP induction accelerated the degradation of NLS-d1EGFP. When both inhibitors were present, NLS-d1EGFP levels remained unaffected by LTP induction. Moreover, LTP-induced acceleration of NLS-d1EGFP synthesis was blocked by rapamycin, which is consistent with the involvement of dendritic mammalian target of rapamycin in LTP-triggered translational activity. Our results clearly demonstrate that LTP induction not only leads to a rapid increase in the rate of protein synthesis but also accelerates protein degradation via the proteasome system.

Efficient expansion of HIV-1-specific T cell responses by homologous immunization with recombinant Semliki Forest virus particles

Vaccines based on recombinant viruses represent a promising strategy for the development of a prophylactic vaccine against HIV-1. However, despite a proven capacity to stimulate potent HIV-1-specific immune responses, viral systems have limited utility in homologous prime-boost regimens due to the generation of anti-vector immune responses. It is therefore important to develop a diverse set of vaccine candidates that can be combined in different heterologous prime-boost regimens and/or to identify a vaccine candidate that is less sensitive to anti-vector mediated immunity. In this report, we describe the design and pre-clinical immunogenicity of a Semliki Forest virus-based vaccine, VREP-C, encoding Indian origin HIV-1 clade C antigens. We show that a single immunization with VREP-C stimulates HIV-1-specific IFNγ ELISPOT responses, which were efficiently boosted by a second and a third homologous VREP-C immunization resulting in highly potent cytotoxic T cell responses. These results suggest that VREP-C may be a valuable component of a future prophylactic vaccine against HIV-1.

Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.

An RNA Vaccine Based on Recombinant Semliki Forest Virus Particles Expressing the Cu,Zn Superoxide Dismutase Protein of Brucella abortus Induces Protective Immunity in BALB/c Mice

We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.

DNA immunization followed by a viral vector booster in a Chlamydia pneumoniae mouse model

Vaccination against Chlamydia pneumoniae would be a beneficial strategy for either preventing or controlling infection by this human respiratory pathogen that also causes persistent infections. In the present study, we used recombinant Semliki Forest virus (rSFV) particles for delivering C. pneumoniae antigens major outer membrane protein (MOMP) or outer membrane protein 2 (Omp2) to the mice or applied the prime-boost technique, where mice were first primed with naked DNA and then boosted with the viral vector coding for the same proteins. Partial protection suggested by the reduced number of cultivable bacteria from the lungs of the challenged mice was seen in mice immunized by either method with MOMP expressing constructs. A significant protection was also achieved after DNA/rSFV immunization with Omp2. DNA priming followed by rSFV boosting induced a more prominent IFN-γ production after challenge at the site of the infection in pulmonary and mediastinal cells.

Multistep regulation of membrane insertion of the fusion peptide of Semliki Forest virus.

A prevailing model for virus membrane fusion proteins has been that the hydrophobic fusion peptide is hidden in the prefusion conformation, becomes exposed once the fusion reaction is triggered, and then either inserts into target membranes or is rapidly inactivated. This model is in general agreement with the structure and mechanism of class I fusion proteins, such as the influenza virus hemagglutinin. We here describe studies of the class II fusion protein E1 from the alphavirus Semliki Forest virus (SFV). SFV fusion is triggered by low pH, which releases E1 from its heterodimeric interaction with the E2 protein and induces the formation of a stable E1 homotrimer. The exposure and target membrane interaction of the E1 fusion peptide (residues 83 to 100) were followed using a monoclonal antibody (MAb E1f) mapping to E1 residues 85 to 95. In agreement with the known structure of SFV and other alphaviruses, the fusion peptide was shielded in native SFV particles and exposed when E1-E2 dimer dissociation was triggered by acidic pH. In contrast, the fusion peptide on purified E1 ectodomains (E1(*)) was fully accessible at neutral pH. Functional assays showed that MAb E1f binding at neutral pH prevented subsequent low-pH-triggered E1(*) interaction with target membranes and trimerization. E1(*) was not inactivated by low pH when treated either in the absence of target membranes or in the presence of fusion-inactive cholesterol-deficient liposomes. Thus, the membrane insertion of the E1 fusion peptide is regulated by additional low-pH-dependent steps after exposure, perhaps involving an E1-cholesterol interaction.

Vesicular stomatitis virus glycoprotein: a transducing coat for SFV-based RNA vectors.

Semliki Forest virus (SFV) vectors have a great potential for the induction of protective immunity in a large number of clinical conditions including cancer. Such a potential accounts for the huge efforts made to improve the in vivo expression from SFV vectors. It is noteworthy that efficient in vivo expression strongly relies on the ability to deliver high-titre vectors. To achieve this, the generation of recombinant SFV particles, using independent expression systems for structural SFV genes, has been proposed. However, despite several modifications in the production process, a risk of contamination with replication-competent, or partially recombined, virus has remained. Here, we exploit the ability of the vesicular stomatitis virus glycoprotein (VSV-G), expressed in trans, to hijack full-length genomic SFV RNA into secreted virus-like particles (VLPs). To allow SFV vector mobilisation, we designed a CMV driven SFV vector in which the internal 26S promoter has been extensively mutated. With this vector, mobilisation events were monitored using the Green Fluorescent Protein (GFP). The production procedure involves a sequential transfection protocol, of plasmids expressing the VSV-G and the SFV vector respectively. We show that the VLPs are effective for cellular delivery of SFV vectors in a broad range of human and non-human cellular targets. Furthermore, production of VLPs is easy and allows, through concentration, the harvest of high-titre vector. The present paper describes a convenient process aimed at mobilising full length SFV vectors. A major issue to consider, while developing clinically relevant gene transfer vectors, is the risk of undesirable generation of replication competent by-products. Importantly, as the VSV-G gene shares no homology with the SFV genome, our VLPs offer a strong guarantee of biosafety.

Induction of P815 tumor immunity by DNA-based recombinant Semliki Forest virus or replicon DNA expressing the P1A gene

Aim: To compare the prophylactic and therapeutic effects of alphaviruses in the same tumor model, we used a DNA-based approach to generate a replicon DNA and recombinant Semliki Forest virus (rSFV) particles expressing P1A, the P815 mastocytoma tumor associated antigen, and compared the immune effect of each vaccine. Methods: Six to eight-week-old female DBA/2 mice were inoculated with P1A plasmid or viral vaccines. Spleen cells were assayed for antigen-specific cytotoxic T cell activity. Tumor growth or survival rate was observed in preventive and therapeutic experiments, respectively. Results: We found that the rSFV particles prevented tumor growth when delivered prior to innoculation of mice with P815 cells, and more importantly, improved survival when delivered after the initiation of tumor growth. Naked P1A replicon DNA also functioned as a protective and therapeutic vaccine, although with less potency than rSFV particles. Virus particles also elicited a stronger cellular immune response as measured by target cell lysis. Conclusion: rSFV particles have stronger specific prophylactic and therapeutic immune effects in mice than replicon DNA-based DNA vaccines, though the latter is more effective than traditional plasmid vectors (e.g. pCI-neo vector).