Recombinant Semliki Forest Virus Research Articles

Semliki Forest Virus (SFV) Self-Amplifying RNA Delivered to J774A.1 Macrophage Lineage by Its Association with a Purified Recombinant SFV Capsid Protein.

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein.

A high throughput antiviral screening platform for alphaviruses based on Semliki Forest virus expressing eGFP reporter gene

Alphaviruses, which contain a variety of mosquito-borne pathogens, are important pathogens of emerging/re-emerging infectious diseases and potential biological weapons. Currently, no specific antiviral drugs are available for the treatment of alphaviruses infection. For most highly pathogenic alphaviruses are classified as risk group-3 agents, the requirement of biosafety level 3 (BSL-3) facilities limits the live virus-based antiviral study. To facilitate the antiviral development of alphaviruses, we developed a high throughput screening (HTS) platform based on a recombinant Semliki Forest virus (SFV) which can be manipulated in BSL-2 laboratory. Using the reverse genetics approach, the recombinant SFV and SFV reporter virus expressing eGFP (SFV-eGFP) were successfully rescued. The SFV-eGFP reporter virus exhibited robust eGFP expression and remained relatively stable after four passages in BHK-21 ​cells. Using a broad-spectrum alphavirus inhibitor ribavirin, we demonstrated that the SFV-eGFP can be used as an effective tool for antiviral study. The SFV-eGFP reporter virus-based HTS assay in a 96-well format was then established and optimized with a robust Z′ score. A section of reference compounds that inhibit highly pathogenic alphaviruses were used to validate that the SFV-eGFP reporter virus-based HTS assay enables rapid screening of potent broad-spectrum inhibitors of alphaviruses. This assay provides a safe and convenient platform for antiviral study of alphaviruses.

GMP manufacturing of Vvax001, a therapeutic anti-HPV vaccine based on recombinant viral particles

Therapeutic vaccination is being explored as a treatment strategy for the treatment of patients with primary or metastatic tumours. We developed a vaccine targeted to Human papillomavirus (HPV)-induced tumours based on recombinant Semliki Forest virus (rSFV) encoding a fusion protein of the E6 and E7 proteins of HPV type 16. To enable a phase I clinical trial with this vaccine, Vvax001, a Good Manufacturing Practice (GMP)-compliant manufacturing process was set up and clinical material was produced.Upstream production of the clinical material resulted in viral titers from 2.4 × 107 to 1.3 × 109 infectious particles/ mL in the harvest. The total volume of 6.0 liter crude virus was purified in 13 consecutive downstream purification runs. The mean titer after purification was 4.0 × 108 infectious particles/ mL and the mean recovery was 19%. Finally, clinical material was filled at a target concentration of 1.25 × 108 infectious particles/mL. Release testing included tests for viral titer and virus identity, biological activity, sterility, bacterial endotoxins, adventitious viruses and absence of replication competent virus. The product complied with all specifications and was released for use as an investigational medicinal product.This is the first GMP production process developed for a SFV-based therapeutic vaccine. The vaccine, Vvax001 is targeted to HPV and has shown promising results in preclinical studies. The GMP-produced Vvax001 material met the quality criteria and was of sufficient quantity to enable assessment of its immunogenicity, safety and efficacy in a clinical setting.

Lack of expression of hepatitis C virus core protein in human monocyte-erived dendritic cells using recombinant semliki forest virus

Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions.

An optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV)

The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 μg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 μg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 μg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 μg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.

Alphavirus-based hepatitis C virus therapeutic vaccines: can universal helper epitopes enhance HCV-specific cytotoxic T lymphocyte responses?

Background:Antigen-specific T cell immune responses play a pivotal role in resolving acute and chronic hepatitis C virus (HCV) infections. Currently, no prophylactic or therapeutic vaccines against HCV are available. We previously demonstrated the preclinical potency of therapeutic HCV vaccines based on recombinant Semliki Forest virus (SFV) replicon particles. However, clinical trials do not always meet the high expectations of preclinical studies, thus, optimization of vaccine strategies is crucial. In efforts to further increase the frequency of HCV-specific immune responses in the candidate SFV-based vaccines, the authors assessed whether inclusion of three strong, so-called universal helper T cell epitopes, and an endoplasmic reticulum localization, and retention signal (collectively termed sigHELP-KDEL cassette) could enhance HCV-specific immune responses.Methods:We included the sigHELP-KDEL cassette in two of the candidate SFV-based HCV vaccines, targeting NS3/4A and NS5A/B proteins. We characterized the new constructs in vitro for the expression and stability of the transgene-encoded proteins. Their immune efficacy with respect to HCV-specific immune responses in vivo was compared with the parental SFV vaccine expressing the corresponding HCV antigen. Further characterization of the functionality of the HCV-specific CD8+ T cells was assessed by surface and intracellular cytokine staining and flow cytometry analysis.Results:Moderate, but significantly, enhanced frequencies of antigen-specific immune responses were achieved upon lower/suboptimal dosage immunization. In optimal dosage immunization, the inclusion of the cassette did not further increase the frequencies of HCV-specific CD8+ T cells when compared with the parental vaccines and the frequencies of effector and memory populations were identical.Conclusion:We hypothesize that the additional effect of the sigHELP-KDEL cassette in SFV-based vaccines depends on the immunogenicity, nature, and stability of the target antigen expressed by the vaccine.

Intratumoral Immunotherapy with XCL1 and sFlt3L Encoded in Recombinant Semliki Forest Virus-Derived Vectors Fosters Dendritic Cell-Mediated T-cell Cross-Priming.

: Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)-based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38- and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti-PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8β T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNAR-deficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltrating CD8 T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF-treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion. SIGNIFICANCE: These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates cross-priming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion.

Immunization with a suicidal DNA vaccine expressing the E glycoprotein protects ducklings against duck Tembusu virus

BackgroudDuck Tembusu virus (DTMUV), a pathogenic flavivirus, emerged in China since 2010 and causing huge economic loss in the Chinese poultry industry. Although several vaccines have been reported to control DTMUV disease, few effective vaccines are available and new outbreaks were continuously reported. Thus, it is urgently to develop a new effective vaccine for prevention of this disease.MethodsIn this study, a suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon and DTMUV E glycoprotein gene was constructed and the efficacy of this new vaccine was assessed according to humoral and cell-mediated immune responses as well as protection against the DTMUV challenge in ducklings.ResultsOur results showed that the recombinant SFV replicon highly expressed E glycoprotein in DEF cells. After intramuscular injection of this new DNA vaccine in ducklings, robust humoral and cellular immune responses were observed in all immunized ducklings. Moreover, all ducklings were protected against challenge with the virulent DTMUV AH-F10 strain.ConclusionsIn conclusion, we demonstrate that this suicidal DNA vaccine is a promising candidate facilitating the prevention of DTMUV infection.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice.

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

Generation and Functional In Vitro Analysis of Semliki Forest Virus Vectors Encoding TNF-α and IFN-γ.

Cytokine gene delivery by viral vectors is a promising novel strategy for cancer immunotherapy. Semliki Forest virus (SFV) has many advantages as a delivery vector, including the ability to (i) induce p53-independent killing of tumor cells via apoptosis, (ii) elicit a type-I interferon (IFN) response, and (iii) express high levels of the transgene. SFV vectors encoding cytokines such as interleukin (IL)-12 have shown promising therapeutic responses in experimental tumor models. Here, we developed two new recombinant SFV vectors encoding either murine tumor necrosis factor-α (TNF-α) or murine interferon-γ (IFN-γ), two cytokines with documented immunostimulatory and antitumor activity. The SFV vector showed high infection rate and cytotoxicity in mouse and human lung carcinoma cells in vitro. By contrast, mouse and human macrophages were resistant to infection with SFV. The recombinant SFV vectors directly inhibited mouse lung carcinoma cell growth in vitro, while exploiting the cancer cells for production of SFV vector-encoded cytokines. The functionality of SFV vector-derived TNF-α was confirmed through successful induction of cell death in TNF-α-sensitive fibroblasts in a concentration-dependent manner. SFV vector-derived IFN-γ activated macrophages toward a tumoricidal phenotype leading to suppressed Lewis lung carcinoma cell growth in vitro in a concentration-dependent manner. The ability of SFV to provide functional cytokines and infect tumor cells but not macrophages suggests that SFV may be very useful for cancer immunotherapy employing tumor-infiltrating macrophages.

Immunogenicity of virus-like Semliki Forest virus replicon particles expressing Indian HIV-1C gag, env and polRT genes

Development of a vaccine targeting human immunodeficiency virus-1 subtype C (HIV-1C) is an important public health priority in regions with a high prevalence of the clade C virus. The present study demonstrates the immunogenicity of recombinant Semliki Forest virus (SFV)-based virus-like replicon particles (VRPs) expressing Indian HIV-1C env/gag/polRT genes. Immunization of mice with recombinant VRPs in a homologous prime-boost protocol, either individually or in combination, elicited significant antigen-specific IFN-γ T cell responses as detected by the ELISPOT assay. Additionally, Gag-specific TNF-α secreting CD8+ and CD4+ T cells and Env-specific IL-2 secreting T cells were also elicited by mice immunized with Gag and Env constructs, respectively, as estimated by intracellular cytokine staining assay. Moreover, an HIV Pol-specific TNF-α response was elicited in mice immunized with a combination of the three VRP constructs. Furthermore, HIV-1C Gag and Env-specific binding antibodies were elicited as verified by gp120 ELISA and p24 Gag ELISA, respectively. The immunogenicity of VRPs was found to be higher as compared to that of RNA replicons and VRPs may therefore be promising preventive and therapeutic candidate vaccines for the control and management of HIV/AIDS.

Antigen design enhances the immunogenicity of Semliki Forest virus-based therapeutic human papillomavirus vaccines.

Cellular immunity against cancer can be achieved with viral vector- and DNA-based immunizations. In preclinical studies, cancer vaccines are very potent, but in clinical trials these potencies are not achieved yet. Thus, a rational approach to improve cancer vaccines is warranted. We previously demonstrated that the relatively low intrinsic immunogenicity of DNA vaccines could be enhanced by inclusion of endoplasmic reticulum (ER) targeting and universal helper epitopes within the vaccine. We now evaluated whether an optimal antigen format, as defined in DNA vaccines, can further enhance the effectiveness of recombinant Semliki Forest virus (rSFV) vaccines. To this purpose, we generated, characterized and evaluated the efficacy of rSFV replicon particles expressing human papillomavirus E6 and/or E7 proteins fused to several helper T-cell epitopes and an ER targeting signal. Here, we show that inclusion of a helper cassette and an ER targeting signal enhanced protein stability and markedly augmented the frequencies of human papillomavirus-specific T cells. Even at an immunization dose of as low as 10(5) replicon particles, this novel vaccine achieved tumor regression and protection. Thus, even highly effective viral vector vaccines can benefit from an improved antigen format, based on the inclusion of defined helper epitopes and ER targeting.

Tattoo Delivery of a Semliki Forest Virus-Based Vaccine Encoding Human Papillomavirus E6 and E7

The skin is an attractive organ for immunization because of the presence of antigen-presenting cells. Intradermal delivery via tattooing has demonstrated superior vaccine immunogenicity of DNA vaccines in comparison to conventional delivery methods. In this study, we explored the efficacy of tattoo injection of a tumor vaccine based on recombinant Semliki Forest virus replicon particles (rSFV) targeting human papillomavirus (HPV). Tattoo injection of rSFV particles resulted in antigen expression in both the skin and draining lymph nodes. In comparison with intramuscular injection, the overall antigen expression determined at the site of administration and draining lymph nodes was 10-fold lower upon tattoo injection. Delivery of SFV particles encoding the E6 and E7 antigens of human papillomavirus type 16 (SFVeE6,7) via tattooing resulted in HPV-specific cytotoxic T cells and in vivo therapeutic antitumor response. Strikingly, despite the observed lower overall transgene expression, SFVeE6,7 delivered via tattoo injection resulted in higher or equal levels of immune responses as compared to intramuscular injection. The intrinsic immunogenic potential of tattooing provides a benefit for immunotherapy based on an alphavirus.

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.

Alphavirus-based Vaccines Encoding Nonstructural Proteins of Hepatitis C Virus Induce Robust and Protective T-cell Responses

An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all- or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

Short-Term Intratumoral Interleukin-12 Expressed from an Alphaviral Vector Is Sufficient to Induce an Efficient Antitumoral Response Against Spontaneous Hepatocellular Carcinomas

Interleukin-12 (IL-12) is an immunostimulatory cytokine that has shown strong antitumor effects in animal models of liver cancer. In order to overcome the severe toxicity associated with its systemic administration, we had previously tested different strategies based on IL-12 gene transfer to tumor cells or to the surrounding liver tissue. We obtained promising results both with a recombinant Semliki Forest virus (SFV) vector expressing high levels of IL-12 (SFV-IL-12) after intratumoral injection and with a plasmid vector [pTonL2(T)-mIL12] that allows liver-specific and inducible IL-12 expression. The aim of the present study was to compare the antitumor responses induced by both systems in a clinically relevant animal model of hepatocellular carcinoma (HCC) developed in L-PK/c-myc transgenic mice. These animals overexpress the c-myc oncogene in their livers, giving rise to spontaneous hepatic tumors with latency, histopathology, and genetic characteristics similar to human HCCs. We observed that intratumoral inoculation of SFV-IL-12 induced growth arrest in most tumors, providing 100% survival rate, in contrast to no survival in control animals. Similar results were obtained with hydrodynamic injection of pTonL2(T)-mIL12 after long-term induction of IL-12 expression in the liver. However, tumor arrest was less evident in plasmid-treated mice and the survival rate was slightly lower, despite higher and more sustained levels of IL-12 and IFN-γ in serum. The fact that SFV-IL-12 was able to induce both apoptosis and a type-I IFN response specifically in the tumor could explain why short-term IL-12 expression from this vector was sufficient to mediate an antitumoral response comparable with long-term IL-12 expression driven by pTonL2(T)-mIL12. Since SFV-IL-12 could reduce the possible toxicity associated with long-term IL-12 expression, we believe that this vector could have a potential application for HCC gene therapy.

Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus

Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SFV RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses.

Enhanced potency of replicon vaccine using one vector to simultaneously co-express antigen and interleukin-4 molecular adjuvant

We evaluated the utility of interleukin-4 (IL-4) as molecular adjuvant of replicon vaccines for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In both Balb/c and C57/BL6 mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) encoding the Hc gene of BoNT/A (AHc), the immunogenicity was significantly modulated and enhanced by co-delivery or co-express of the IL-4 molecular adjuvant. The enhanced potencies were also produced by co-delivery or co-expression of the IL-4 molecular adjuvant in mice immunized with the recombinant SFV replicon particles (VRP) vaccines. In particular, when AHc and IL-4 were co-expressed within the same replicon vaccine vector using dual-expression or bicistronic IRES, the anti-AHc antibody titers, serum neutralization titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased. These results indicate IL-4 is an effective Th2-type adjuvant for the replicon vaccines in both strain mice, and the co-expression replicon vaccines described here may be an excellent candidate for further vaccine development in other animals or humans. Thus, we described a strategy to design and develop efficient vaccines against BoNT/A or other pathogens using one replicon vector to simultaneously co-express antigen and molecular adjuvant.

Phenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.

Reversible stress-induced lipid body formation in fast twitch rat myofibers

We analyzed the existence of lipid bodies (LBs) in the fast twitch rat flexor digitorum brevis (FDB) myofibers and found that these structures were scarce. However, isolation procedure of the myofibers, heath shock, viral infection or the glycosylation inhibitor tunicamycin induced formation of the LBs, which were stationary structures flanking Z lines. We next infected FDB myofibers with recombinant Semliki Forest virus expressing caveolin 3-yellow fluorescent protein (cav3-YFP) since this chimeric protein was targeted to the LBs facilitating their further analysis. Photobleaching experiments showed that the LBs recovered cav 3-YFP extremely slowly, indicating that they were not continuous with the endoplasmic/sarcoplasmic reticulum. We found, however, that cav3-YFP could move from the LBs to the sarcolemma and this phenomenon was sensitive to Brefeldin A, suggesting that the chimeric protein could be returned from the LBs to the endoplasmic reticulum.