A Semliki forest virus vector engineered to express IFNα induces efficient elimination of established tumors.

Semliki Forest virus (SFV) represents a promising gene therapy vector for tumor treatment, because it produces high levels of recombinant therapeutic proteins while inducing apoptosis in infected cells. In this study, we constructed a SFV vector expressing murine interferon alpha (IFNα). IFNα displays antitumor activity mainly by enhancing an antitumor immune response, as well as by a direct antiproliferative effect. In spite of the antiviral activity of IFNα, SFV-IFN could be produced in BHK cells at high titers. This vector was able to infect TC-1 cells, a tumor cell line expressing E6 and E7 proteins of human papillomavirus, leading to high production of IFNα both in vitro and in vivo. When injected into subcutaneous TC-1 tumors implanted in mice, SFV-IFN was able to induce an E7-specific cytotoxic T lymphocyte response, and to modify tumor infiltrating immune cells, reducing the percentage of T regulatory cells and activating myeloid cells. As a consequence, SFV-IFN was able to eradicate 58% of established tumors treated 21 days after implantation with long-term tumor-free survival and very low toxicity. SFV-IFN was also able to induce significant antitumor responses in a subcutaneous tumor model of murine colon adenocarcimoma. These data suggest that local production of IFNα by intratumoral injection of recombinant SFV-IFN could represent a potent new strategy to treat tumors in patients.

Enhancing immune responses using suicidal DNA vaccines.

We describe a DNA vaccine strategy that allows antigens to be produced in vivo in the context of an alphaviral replicon. Mice immunized with such vectors developed humoral and cellular immune responses at higher levels than mice that received a conventional DNA vaccine vector. Immunized animals acquired protective immunity to lethal influenza challenge. Compared with traditional DNA vaccine strategies in which vectors are persistent and the expression constitutive, the expression mediated by the alphaviral vector was transient and lytic. As a result, biosafety risks such as chromosomal integration, and the induction of immunological tolerance, could be circumvented.

Cytokine gene transfer into dendritic cells for cancer treatment.

Bone marrow-derived dendritic cells have been used to treat established experimental tumors by unleashing a cellular immune response against tumor antigens. Such antigens are artificially loaded onto dendritic cells' antigen-presenting molecules by different techniques including incubation with synthetic antigenic determinants, tumor lysates or nucleic acids encoding for those relevant antigens. Ex vivo gene transfer with viral and non-viral vectors is frequently used to obtain expression of the tumor antigens and thereby to formulate the therapeutic vaccines. Efficacy of the approaches is greatly enhanced if dendritic cells are transfected with a number of genes which encode immunostimulating factors. In some cases, such as with IL-12, IL-7 and CD40L genes, injection inside experimental malignancies of thus transfected dendritic cells induces complete tumor regression in several models. In this case tumor antigens are captured by dendritic cells by still unclear mechanisms and transported to lymphoid organs where productive antigen presentation to T-cells takes place. Many clinical trials testing dendritic cell-based vaccines against cancer are in progress and partial clinical efficacy has been already proved. Transfection of genes further strengthening the immunogenicity of such strategies will join the clinical club soon.

A continuous epitope from transmissible gastroenteritis virus S protein fused to E. coli heat-labile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity.

Antigenic site D from the spike protein of transmissible gastroenteritis virus (TGEV), which is a continuous epitope critical in neutralization, has been expressed as a fusion protein with E. coli heat-labile toxin B subunit (LT-B) in attenuated S. typhimurium. Synthetic peptides containing the sequence of site D induced TGEV neutralizing antibodies when inoculated subcutaneously in both rabbits and swine. A synthetic oligonucleotide encoding residues 373-398 of TGEV S protein, including antigenic site D, was cloned in frame with the 3' end of LT-B gene, into a plasmid used to transform S. typhimurium delta asd chi 3730. A collection of 6 recombinant plasmids designated pYALTB-D I-VI encoding LTB-site D fusions with a variable number of site D sequences were selected. Four of the 6 LTB-site D fusion products expressed in S. typhimurium chi 3730 formed oligomers (pentamers) that dissociated at > 70 degrees. S. typhimurium chi 3730 (pYALTB-D) V and VI expressed the oligomer forming products with higher antigenicity. Partially purified LTB-site D fusion product expressed from S. typhimurium chi 3730 (pYALTB-D) V induced anti-TGEV neutralizing antibodies in rabbits. Recombinant vaccine strain S. typhimurium delta cya delta crp delta asd chi 3987 transformed with plasmid pYALTB-D V expressed constitutively products that formed oligomers presumably containing 20 copies of site D, and showed a high stability in vitro. This recombinant strain was orally inoculated in rabbits and induced TGEV specific antibodies in both serum and intestinal secretion.

Non-viral amplification systems for gene transfer: vectors based on alphaviruses.

Non-viral self-replicating vectors based on defective viral genomes have been developed for a number of different alphaviruses including Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE). These vectors can be used for gene delivery as naked RNA or DNA. Recombinant alphavirus RNA can be synthesized in vitro from plasmids containing the alphavirus replicon under the control of a prokaryotic promoter such as SP6 or T7. These self-replicating RNAs have been able to induce protective immune responses in vivo, probably due to the high level of expression of the recombinant antigen in the transfected cells. However, alphavirus vectors based on the direct delivery DNA are probably a better choice due to their higher stability and lower production cost. In these vectors, the alphavirus replicon is placed under the control of a RNA polymerase II promoter. These vectors are more efficient than conventional plasmids in inducing both humoral and cellular immune responses in small animals, allowing the use of significant smaller amounts of DNA for immunization. In addition, due to the transient nature of the alphavirus replicons, possible problems associated with DNA integration into host chromosomes are eliminated.

Characterization of transmissible gastroenteritis coronavirus S protein expression products in avirulent S. typhimurium delta cya delta crp: persistence, stability and immune response in swine.

The spike protein from transmissible gastroenteritis virus (TGEV) was expressed in attenuated S. typhimurium delta cya delta crp delta asd chi 3987. Three partially overlapping fragments of TGEV S gene, encoding the amino-terminal, intermediate, and carboxy-terminal end of the protein, as well as the full length gene were inserted into the asd+ plasmid pYA292 to generate recombinant plasmids pYATS-1, pYATS-2, pYATS-3, and pYATS-4, respectively, which were transformed into S. typhimurium chi 3987. Recombinant S. typhimurium chi 3987 (pYATS-1) and chi 3987 (pYATS-4) expressing constitutively a 53 kDa amino-terminal fragment of the S protein and the full length protein (144 kDa), respectively, showed high stability. After 50 generations in vitro 60% and 20% of the bacteria transformed with pYATS-1 and pYATS-4, respectively, expressed the S-protein antigen. Since S. typhimurium chi 3987 (pYATS-1) showed a better level of expression and stability in vitro, this recombinant strain was selected as a potential bivalent vector to induce both immunity to Salmonella and TGEV in swine. In order to study colonization of swine tissues by S. typhimurium delta cya delta crp, a gene conferring resistance to rifampicin was cloned into the chromosome of S. typhimurium chi 3987, generating chi 4509 strain. Both S. typhimurium chi 4509 (pYA292) and chi 4509 (pYATS-1) colonized the ileum of orally inoculated swine with clearance of bacteria between days 10-20 post-infection. The expression of the amino-terminal fragment of the S protein diminished the ability of S. typhimurium chi 4509 (pYATS-1) to colonize deep tissues. The recombinant strain S. typhimurium chi 3987 (pYATS-1) induced TGEV specific antibodies in both serum and saliva of orally inoculated swine.

Antigen selection and presentation to protect against transmissible gastroenteritis coronavirus.

The antigenic structure of the S glycoprotein of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) has been determined and correlated with the physical structure. Four antigenic sites have been defined (A, B, C, and D). The sites involved in the neutralization of TGEV are: A, D, and B, sites A and D being antigenically dominant for TGEV neutralization in vitro. These two sites have specific properties of interest: site A is highly conserved and is present in coronaviruses of three animal species, and site D can be represented by synthetic peptides. Both sites might be relevant in protection in vivo. PRCV does not have sites B and C, due to a genomic deletion. Complex antigenic sites, i.e., conformation and glycosylation dependent sites, have been represented by simple mimotopes selected from a library expressing recombinant peptides with random sequences, or by anti-idiotypic internal image monoclonal antibodies. An epidemiological tree relating the TGEVs and PRCVs has been proposed. The estimated mutation fixation rate of 7 +/- 2 x 10(-4) substitutions per nucleotide and year indicates that TGEV related coronaviruses show similar variability to other RNA viruses. In order to induce secretory immunity, different segments of the S gene have been expressed using a virulent forms of Salmonella typhimurium and adenovirus. These vectors, with a tropism for Peyer's patches may be ideal candidates in protection against TGEV.

Structure and encapsidation of transmissible gastroenteritis coronavirus (TGEV) defective interfering genomes.

Serial undiluted passages were performed with the PUR46 strain of TGEV in swine testis (ST) cells. Total cellular RNA was analyzed at different passages after orthophosphate metabolic labeling. Three new defective RNA species of 24, 10.5, and 9.5 kb (DI-A, DI-B, and DI-C respectively) were detected at passage 30, which were highly stable and significantly interfered with helper mRNA synthesis in subsequent passages. By Northern hybridization DIs A, B, and C were detected in purified virions at amounts similar to those of helper RNA. Standard and defective TGEV virions could be sorted in sucrose gradients, indicating that defective and full-length genomes are independently packaged. cDNA synthesis of DI-B and DI-C RNAs was performed by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments in each case. Cloning and sequencing of the DI-C PCR products showed that the smallest DI particle comprises 9.5 kb and has 4 discontinuous regions of the genome. It contains 2.1 kb from the 5'-end of the genome, about 7 kb from gene 1b, the first 24 nucleotides of the S gene, 12 nucleotides of ORF 7, and the 0.4 kb of the UTR at the 3'-end.

The spike protein of transmissible gastroenteritis coronavirus controls the tropism of pseudorecombinant virions engineered using synthetic minigenomes.

The minimum sequence required for the replication and packaging of transmissible gastroenteritis virus (TGEV)-derived minigenomes has been determined. To this end, cDNAs encoding defective RNAs have been cloned and used to express heterologous spike proteins, to determine the influence of the peplomer protein in the control of TGEV tropism. A TGEV defective interfering RNA of 9.7 kb (DI-C) was isolated, and a cDNA complementary to DI-C RNA was cloned under the control of T7 promoter. In vitro transcribed DI-C RNA was replicated in trans upon transfection of helper virus-infected cells. A collection of DI-C deletion mutants (TGEV minigenomes) was generated and tested for their ability to be replicated and packaged. The size of the smallest minigenome replicated in trans was 3.3 kb. The rescue system was used to express the spike protein of an enteric TGEV isolate (C11) using as helper virus a TGEV strain (C8) that replicates very little in the gut. A mixture of two pseudorecombinant viruses containing either the helper virus genome or the minigenome was obtained. These pseudorecombinants display in the surface the S proteins from the enteric and the attenuated virus, and showed 10(4)-fold increase in their gut replication levels as compared to the helper isolate (C8). In addition, the pseudorecombinant virus increased its enteric pathogenicity as compared to the C8 isolate.

Two-helper RNA system for production of recombinant Semliki forest virus particles.

Alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. However, present strategies for production of such particles have biosafety limitations due to the generation, by RNA recombination, of replication-proficient viruses (RPVs). Here we describe a new packaging system for Semliki Forest virus (SFV) based on a the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 polyprotein are expressed from two independent RNA molecules. The capsid gene contains a translational enhancer and therefore that sequence was also engineered in front of the spike sequence p62-6K-E1. A sequence coding for the foot-and-mouth disease virus 2A autoprotease was inserted in frame between the capsid translational enhancer and the spike genes. This allows production of the spike proteins at high levels with cotranslational removal of the enhancer sequence and normal biosynthesis of the spike complex. The autoprotease activity of the capsid protein was abolished by mutation, further increasing the biosafety of the system. Cotransfection of cells with both helper RNAs and an SFV vector replicon carrying the LacZ gene led to production of recombinant particles with titers of up to 8 x 10(8) particles per 10(6) cells. Extensive analysis failed to demonstrate the presence of any RPVs, emphasizing the high biosafety of the system based on two-helper RNAs.

Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice.

The replicon of Semliki Forest virus (SFV) offers the possibility to direct high-level, transient expression of heterologous proteins in vivo. We initiated studies to determine the possibility of employing the SFV expression system for recombinant vaccine purposes. Mice immunized with recombinant SFV encoding Influenza A nucleoprotein (NP) or E. coli LacZ developed long-lasting antigen-specific IgG levels and induction of cytotoxic T-cell (CTL) memory that persisted for over one year. Predominantly type 1 T-helper cells were induced as shown by IgG subclass ELISA. Humoral and cell-mediated immune responses could be induced upon delivery by several administration routes and mucosal immunizations induced secretory IgA in the respiratory tract. Development of immune responses against the vector itself did not inhibit boost responses by subsequent immunizations with recombinant SFV. Immunization of mice with vectors encoding the Influenza A virus antigens nucleoprotein (NP) and hemagglutinin (HA) resulted in immune responses that were protective against challenge infection with Influenza virus.

Molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity.

Three transmissible gastroenteritis virus (TGEV) defective RNAs were selected by serial undiluted passage of the PUR46 strain in ST cells. These RNAs of 22, 10.6, and 9.7 kb (DI-A, DI-B, and DI-C, respectively) were detected at passage 30, remained stable upon further passage in cell culture, and significantly interfered with helper mRNA synthesis. RNA analysis from purified virions showed that the three defective RNAs were efficiently packaged. Virions of different densities containing either full-length or defective RNAs were sorted in sucrose gradients, indicating that defective and full-length genomes were independently encapsidated. DI-B and DI-C RNAs were amplified by the reverse transcription-polymerase chain reaction, cloned, and sequenced. DI-B and DI-C genomes are formed by three and four discontinuous regions of the wild-type genome, respectively. DI-C contains 2144 nucleotides (nt) from the 5'-end of the genome, two fragments of 4540 and 2531 nt mostly from gene 1b, and 493 nt from the 3' end of the genome. DI-B and DI-C RNAs include sequences with the pseudoknot motif and encoding the polymerase, metal ion binding, and helicase motifs. DI-B RNA has a structure closely related to DI-C RNA with two main differences: it maintains the entire ORF 1b and shows heterogeneity in the size of the 3' end deletion. This heterogeneity maps at the beginning of the S gene, where other natural TGEV recombination events have been observed, suggesting that either a process of template switching occurs with high frequency at this point or that the derived genomes have a selective advantage.

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.

Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes.

The sequences involved in the replication and packaging of transmissible gastroenteritis virus (TGEV) RNA have been studied. The structure of a TGEV defective interfering RNA of 9.7 kb (DI-C) was described previously (A. Mendez, C. Smerdou, A. Izeta, F. Gebauer, and L. Enjuanes, Virology 217: 495-507, 1996), and a cDNA with the information to encode DI-C RNA was cloned under the control of the T7 promoter. The molecularly cloned DI-C RNA was replicated in trans upon transfection of helper virus-infected cells and inhibited 20-fold the replication of the parental genome. A collection of 14 DI-C RNA deletion mutants (TGEV minigenomes) was synthetically generated and tested for their ability to be replicated and packaged. The smallest minigenome (M33) that was replicated by the helper virus and efficiently packaged was 3.3 kb. A minigenome of 2.1 kb (M21) was also replicated, but it was packaged with much lower efficiency than the M33 minigenome, suggesting that it had lost either the sequences containing the main packaging signal or the required secondary structure in the packaging signal due to alteration of the flanking sequences. The low packaging efficiency of the M21 minigenome was not due to minimum size restrictions. The sequences essential for minigenome replication by the helper virus were reduced to 1,348 nt and 492 nt at the 5' and 3' ends, respectively. The TGEV-derived RNA minigenomes were successfully expressed following a two-step amplification system that couples pol II-driven transcription in the nucleus to replication supported by helper virus in the cytoplasm, without any obvious splicing. This system and the use of the reporter gene beta-glucuronidase (GUS) allowed minigenome detection at passage zero, making it possible to distinguish replication efficiency from packaging capability. The synthetic minigenomes have been used to design a helper-dependent expression system that produces around 1.0 microgram/10(6) cells of GUS.

Mechanisms of transmissible gastroenteritis coronavirus neutralization.

Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies of a single specificity [monoclonal antibodies (MAbs)]. Most of the virus was neutralized in the first 2-3 min of a reversible reaction, which was followed by a second phase with a decreased neutralization rate and, in some cases, by a persistent fraction, which was a function of the MAb and of the antibody-to-virus ratio. Neutralization of TGEV is a specific event that requires the location of the epitope involved in the neutralization in the appropriate structural context, which is present in the wild-type virus but not in certain MAb escaping mutants. In neutralization of TGEV by binary combinations of MAbs specific for the same or for different antigenic sites, either no cooperation or a synergistic effect, respectively, was observed. Mechanisms of TGEV neutralization by MAbs were characterized at high, intermediate, and low antibody-to-virus ratios. Under these conditions, mainly three steps of the replication cycle were inhibited: binding of virus to the cell, internalization, and a step that takes place after internalization. In addition, virus aggregation could be responsible for the neutralization of 10 to 20% of virus infectivity.

Induction of antibodies protecting against transmissible gastroenteritis coronavirus (TGEV) by recombinant adenovirus expressing TGEV spike protein.

Ten recombinant adenoviruses expressing either fragments of 1135, 1587, or 3329 nt or the full-length spike gene of transmissible gastroenteritis coronavirus (TGEV) have been constructed. These recombinants produce S polypeptides with apparent molecular masses of 68, 86, 135, and 200 kDa, respectively. Expression of the recombinant antigen driven by Ad5 promoters was inhibited by the insertion of an exogenous SV-40 promoter. Most of the recombinant antigens remain intracytoplasmic in infected cells. All the recombinant-directed expression products contain functional antigenic sites C and B (Gebauer et al., 1991, Virology 183, 225-238). The recombinant antigen of 135 kDa and that of 200 kDa, which represents the whole spike protein, also contain antigenic sites D and A, which have previously been shown to be the major inducers of TGEV-neutralizing antibodies. Interestingly, here we show that recombinant S protein fragments expressing only sites C and B also induced TGEV-neutralizing antibodies. The chimeric Ad5-TGEV recombinants elicited lactogenic immunity in hamsters, including the production of TGEV-neutralizing antibodies. The antisera induced in swine by the Ad5 recombinants expressing the amino-terminal 26% of the spike protein (containing sites C and B) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively.