Alu and LINE-1 (L1), which constitute ~11% and ~17% of the human genome, respectively, are transposable non-LTR retroelements. They transpose not only in germ cells but also in somatic cells, occasionally causing cancer. We have previously demonstrated that antiretroviral restriction factors, human APOBEC3 (hA3) proteins (A–H), differentially inhibit L1 retrotransposition. In this present study, we found that hA3 members also restrict Alu retrotransposition at differential levels that correlate with those observed previously for L1 inhibition. Through deletion analyses based on the best-characterized hA3 member human APOBEC3G (hA3G), its N-terminal 30 amino acids were required for its inhibitory activity against Alu retrotransposition. The inhibitory effect of hA3G on Alu retrotransposition was associated with its oligomerization that was affected by the deletion of its N-terminal 30 amino acids. Through structural modeling, the amino acids 24 to 28 of hA3G were predicted to be located at the interface of the dimer. The mutation of these residues resulted in abrogated hA3G oligomerization, and consistently abolished the inhibitory activity of hA3G against Alu retrotransposition. Importantly, the anti-L1 activity of hA3G was also associated with hA3G oligomerization. These results suggest that the inhibitory activities of hA3G against Alu and L1 retrotransposition might involve a common mechanism. Alu, which comprise ~17% and ~11% of the human genome, respectively [Alu elements do not encode a reverse transcriptase or an endonuclease; rather, the transcribed Alu RNAs hijack the L1-encoded enzymes to move to new locations in the genome through mechanisms that are as yet unclear [Alu occurs not only in germ cells, causing several genetic diseases [Retrotransposons compose ~42% of the human genome, and these elements are classified into the non-LTR and LTR classes. Non-LTR retrotransposons are subdivided into long interspersed elements (LINEs) and short interspersed elements (SINEs), representatives of which are LINE-1 (L1) and  unclear . Importadiseases -13, but diseases ,15, and diseases , breast diseases , and hepdiseases . These fAlu and L1 retrotransposons ([Human APOBEC3G (hA3G) is one of the seven members of the APOBEC3 (hA3) family of cytidine deaminases (hA3A to hA3H). hA3G is known to be an intrinsic retroviral restriction factor that inhibits Vif-defective human immunodeficiency virus type 1 (HIV-1) infection by being incorporated into viral particles and mediating extensive deamination of the nascent minus-strand viral DNA during reverse transcription, which results in G-to-A hypermutation -23. Thissposons . hA3G sposons , Vif-deficient HIV-1 proviral indicator construct pNLLuc-F(-)E(-), and VSV-G expression plasmid pHIT/G have previously been described elsewhere [The hemagglutinin (HA)-tagged hA3 expression plasmids , the GFP expression plasmid pCA-EGFP, the empty expression vector pCAGGS-HA, the L1 indicator construct pCEP4/L1mneoI/ColE1 (kindly provided by N. Gilbert), the L1 ORF2 expression plasmid pBudORF2opt (kindly provided by A.M. Roy-Engel), the lsewhere ,52-55 (nHeLa and 293T cells were maintained under standard conditions. 293T cells were transfected with HA-tagged hA3 wild-type and mutant plasmids using the FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer’s instructions. Cell extracts from transfected cells were subjected to gel electrophoresis and then transferred to a nitrocellulose membrane. The membranes were probed with an anti-HA mouse monoclonal antibody (Sigma). The antibody-bound proteins were visualized to confirm hA3 protein expression by chemiluminescence using an ECL Western blotting detection system  and an LAS-3000 imaging system (FujiFilm).HeLa cells were plated on 13-mm glass coverslips and transfected with 0.5 µg of hA3 expressing plasmids by using FUGENE6. The transfected cells were fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized with 0.05% saponin for 10 min, and immunostained with an anti-HA monoclonal antibody (5 µg/ml). The secondary goat anti-mouse antibody that was conjugated with Cy3 was used at 5 µg/ml. All immunofluorescence images were observed on a Leica DMRB microscope  equipped with a 63×1.32 NA oil immersion lens (PL APO), acquired through a cooled CCD camera, MicroMAX , and digitally processed using IPlab Software . All images were assembled using Adobe Photoshop .Alu retrotransposition assays were performed by co-transfecting 2 x 105 HeLa cells with 0.1 μg of the respective hA3 expression plasmid  together with either 0.3 μg of the neomycin-resistance (neor)-based L1 expression vector pCEP4/L1mneoI/ColE1 and 0.1 μg of an empty vector (for the L1 retrotransposition assay) or 0.3 μg of the neor-based Alu expression vector pYa5neotet and 0.1 μg of the L1 ORF2 expression plasmid pBudORF2opt (for the Alu retrotransposition assay) using Lipofectamine and Plus reagents (Invitrogen). As a negative control, 0.5 μg of a GFP expression vector, pCA-EGFP, was transfected into HeLa cells. After 72 h, the cells were trypsinized, re-seeded into T25 or T75 flasks for G418 selection (1 mg/ml for the L1 assay and 400 μg/ml for the Alu assay), and maintained. At 14 days after selection, the resultant G418-resistant (G418R) colonies were fixed, stained with crystal violet (Merck), and counted.L1 and To perform a coimmunoprecipitation-based oligomerization assay, plasmids (0.5 μg) expressing HA-tagged wild-type and mutant hA3G were transfected along with phA3G-myc (0.5 μg) into 293T cells using FuGENE 6. After 48 h, the transfected cells were suspended in 500 μl of RIPA buffer . The resultant lysates were clarified by brief centrifugation, pre-cleared with 30 μl of Protein A-Agarose Fast Flow  for 1 h at 4°C, and then incubated with an anti-myc affinity gel (Sigma). After 1 h at 4°C, the immune complexes were extensively washed with RIPA buffer. Equal aliquots of the total and bound fractions were subjected to gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were probed with an anti-HA mouse monoclonal antibody (Sigma) or an anti-β-actin mouse monoclonal antibody . The signal intensities of the immunoprecipitated hA3G protein on Western blots were quantified using the LAS-3000 imaging system (Fujifilm). For the RNase A treatment experiment, the immune complexes were separated into two aliquots. The wild-type sample was incubated with or without 25 U of RNase A (Sigma) at room temperature for 30 min. Samples were extensively washed and then resuspended in loading dye. The samples were assayed as described above.Head-to-head dimer models of hA3G N-terminal domain were obtained by homology modelling using either the crystal structure of human APOBEC2 (hA2) at a resolution of 2.50 Å or the NMR structure of the C-terminal domain of hA3G  ,66, and activity . These oAlu retrotransposition was abolished in the mutants carrying an N-terminal deletion of 30 or more residues  NMR structure in parallel. Both structural modeling of wild-type hA3G revealed that, among the N-terminal 30 amino acids, a cluster of dimer interface residues  located in the N-terminal core structure α1-loop-β1 of hA3G interact with the counterpart residues of another monomer . Importaposition . hA3G muposition  and S3B.position . Taken aAlu retrotransposition resembles its effects on L1 retrotransposition in two regards, first, that hA3G showed similar levels of inhibitory activity against the both retrotransposition events , and 4G(124–127), did not inhibit L1 retrotransposition (Alu retrotransposition in Alu retrotransposition is associated with hA3G oligomerization but independent of its deaminase activity. We therefore postulate that the inhibitory activities of hA3G against Alu and L1 retrotransposition might share common mechanism(s). The inhibitory effects of the hA3G protein on n events ,37. Thesposition , and 7C,Alu retrotransposition at differential levels, which are very similar to the levels at which these host proteins block L1 retrotransposition. With respect to hA3G, the N-terminal 30 amino acids are important for the anti-Alu activity. The ability of hA3G to inhibit Alu retrotransposition was independent of its deaminase activity but associated with its oligomerization activity, as previously reported by Hulme et al. [Alu retrotransposition are required for the oligomerization of this protein. We used structural modeling to identify the specific residues among the N-terminal 30 amino acids that are responsible for the oligomerization of hA3G. We finally identified amino acid residues 24–28 of hA3G as the contributors of oligomerization. Our present study demonstrated that hA3 family proteins inhibit e et al.  and Bulle et al. , respectAlu retrotransposition. This hypothesis makes sense because Alu elements do not encode a functional reverse transcriptase or endonuclease, and therefore, they need to hijack the L1-encoded enzymatic machinery for retrotransposition through mechanisms that are currently unclear. It is intriguing to speculate that hA3G might be able to physically block both the Alu and L1 retroelements because hA3G is intrinsically an RNA-binding protein that can associate non-specifically with cellular RNAs [Alu retroelements [Alu reverse transcription, and are dependent on the ability of hA3G to form oligomers. In the former case, Alu RNA per se might help stabilize hA3G oligomer formation, as suggested in Importantly, these residues were also critical for the inhibitory activity of L1 retrotransposon, suggesting that this activity might involve the same mechanism as that of lar RNAs ,59,65,69elements ,70, or bIt was somewhat unexpected to find that the N-terminal 30 amino acids of hA3G are required for oligomerization in our study because amino acid positions 124/127 have previously been reported to be important ,58,59. IAlu elements [Alu retrotransposition, putatively through binding to either the ORF2 protein or Alu RNA as described above.Whereas transcriptional repressors such as SRY, SOX2 and methyl-CpG-binding protein 2 have been reported to negatively regulate L1 retrotransposition at the transcriptional levels -73, postelements ,53. PartAlu and L1 retrotransposition, possibly in cooperation with other cellular factor(s). It should be noted that the superfamily-1 RNA helicase protein MOV10 , whichFigure S1Inhibitory effect of hA3G deletion mutants on HIV-1 infection was evaluated by cotransfecting 293T cells with hA3G and VSV-G plasmids, together with a luciferase-based Vif (-) Env (-) HIV-1 construct, as described by Iwabu et al. . After 48 h, each viral supernatant was harvested. Normalized supernatants were incubated with 293T cells for additional 48 h. Cells were then lysed and subjected to luciferase assay. The data shown are the mean ± SD of triplicate experiments. RLU: relative light units.(TIF)Click here for additional data file.Figure S2Cellular RNA contributes to the stabilization of hA3G’s oligomer. HA-tagged hA3G-WT in the immunoprecipitate as described in (TIF)Click here for additional data file.Figure S3hA3G mutants with individual amino acid substitutions. (A) Oligomerization assay was performed by IP-Western blot analysis, as described in Alu retrotransposition assay was performed as described in R colonies were counted to determine the level of Alu retrotransposition. The data shown are the mean ±SD of triplicate experiments. Mo, mock; WT, wild-type hA3G; GFP, GFP only. *P < 0.05, **P < 0.005, t-test.(TIF)Click here for additional data file.Figure S4Inhibitory effect of hA3G oligomerization mutant proteins on HIV-1 infection. The assay was performed as described in (TIF)Click here for additional data file.


Whole-virus vaccines, including inactivated or live-attenuated influenza vaccines, have been conventionally developed and supported as a prophylaxis. These currently available virus-based influenza vaccines are widely used in the clinic, but the vaccine production takes a long time and a huge number of embryonated chicken eggs. To overcome the imperfection of egg-based influenza vaccines, epitope-based peptide vaccines have been studied as an alternative approach. Here, we formulated an efficacious peptide vaccine without carriers using phosphodiester CpG-DNA and a special liposome complex. Potential epitope peptides predicted from the hemagglutinin (HA) protein of the H5N1 A/Viet Nam/1203/2004 strain  were used to immunize mice along with phosphodiester CpG-DNA co-encapsulated in a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex (Lipoplex(O)) without carriers. We identified a B cell epitope peptide  that can potently induce epitope-specific antibodies. Furthermore, immunization with a complex of the B cell epitope and Lipoplex(O) completely protects mice challenged with a lethal dose of recombinant H5N1 virus. These results suggest that our improved peptide vaccine technology can be promptly applied to vaccine development against pandemic influenza. Furthermore our results suggest that potent epitopes, which cannot be easily found using proteins or a virus as an antigen, can be screened when we use a complex of peptide epitopes and Lipoplex(O). Influenza A viruses have gained attention because pandemic influenza causes human diseases associated with respiratory tract complication and high mortality. Recently, several emerging influenza A subtypes such as H1N1 Diverse studies have endeavored to develop prophylaxis and therapeutics against influenza viruses. Whole-virus vaccines, including inactivated or live-attenuated influenza vaccines, have been conventionally developed and supported as a prophylaxis. Inactivated influenza virus vaccines are safe and can induce significant protective neutralizing antibodies with an efficacy of 60% to 90% In addition, epitope-based peptide vaccines have been used as an alternative approach to improving the immunogenicity, protecting against divergent strains, shortening the production time, and reducing side effects M. bovis genomic DNA Several investigators have reported that CpG-DNA has potent functional effects as a Th1-responsive adjuvant The influenza HA is a surface glycoprotein (HA0), which is cleaved to two polypeptides, HA1 and HA2 When we immunized BALB/c mice with the complex of each epitope from HA1 and Lipoplex(O), the BALB/c mice induced hH5N1 HA58 and hH5N1 HA233 peptide-specific IgG production more markedly than other control mice . The immTo further evaluate the adjuvant activity of Lipoplex(O), we immunized the mice with the complex of previously reported B cell epitopes from HA2 and Lipoplex(O). However, no production of the peptide-specific IgG was observed . ConsideWe compared the effect of each liposome on the IgG production in BALB/c mice after an injection of hH5N1 HA233 peptide and MB-ODN 4531(O) co-encapsulated in different liposomes. The complex of hH5N1 HA233 and MB-ODN 4531(O) co-encapsulated in DOPE:CHEMS induced more robust production of the epitope-specific IgG than the complex of hH5N1 HA233 peptide and MB-ODN 4531(O) co-encapsulated in other liposomes . When weWe compared the efficiency of the epitope-specific IgG production between whole virus immunization and a complex of epitope and Lipoplex(O) immunization in BALB/c mice. We observed a higher increase in the amount of total IgG titer against an inactivated virus in mice immunized with a complex of the inactivated rH5N1 virus and Lipoplex(O) than in mice immunized with hH5N1 HA233 peptide plus Lipoplex(O), free inactivated rH5N1 virus, or a complex of an inactivated rH5N1 virus encapsulated with DOPE:CHEMS . HoweverOf the selected B cell epitopes from the HA protein of the H5N1 A/Vietnam/1203/2004 strain, the hH5N1 HA233 epitope induces the highest epitope-specific antibody production . TherefoTo verify whether the epitope-specific antibodies produced by the complex of epitope and Lipoplex(O) could react with influenza A viruses, we performed a virus neutralization assay. The results were quantified against the recombinant H5N1 A/Vietnam/1203/2004 virus (rH5N1 virus) which was generated by reverse genetics, H1N1 virus (A/WSN/1933 virus), or swine origin H1N1 virus (A/Korea/01/09). The antisera from the mice immunized with the hH5N1 HA233 epitope efficiently neutralized the rH5N1 virus . The croThis outcome indicates that the antisera obtained by hH5N1 HA233 immunization were cross-reactive with the corresponding epitopes of H1N1 influenza strains. It also indicates that the extent of the cross-reactivity depends on amino acid sequences of the specific epitopes.To check whether a vaccine containing a complex of hH5N1 HA233 and Lipoplex(O) has prophylactic effects, we challenged the BALB/c mice with the rH5N1 virus ten days after the vaccination. As a negative control, two groups of mice were immunized with hH5N1 HA233 and MB-ODN 4531GC(O) co-encapsulated in DOPE:CHEMS (LipoplexGC(O)+hH5N1 HA233) or with MB-ODN 4531(O) encapsulated in DOPE:CHEMS (Lipoplex(O)). The mice immunized with a vaccine containing a complex of hH5N1 HA233 and Lipoplex(O) showed a 100% survival rate, whereas all the mice of the negative control groups died . All thePhosphorothioate-modified CpG-DNA (PS-ODN) has been studied for prophylactic application against infectious diseases in mice. Several investigations reported that mice pre-treated with PS-ODN were completely or partially protected against influenza A virus challenge depending on the periods and routes of PS-ODN pre-treatment We next investigated the prophylactic efficacy of a vaccine containing other B cell epitopes (hH1N1-WSN HA233 and hH1N1-HK HA233). The hH1N1-WSN HA233 vaccine was prophylactically effective against the maA/WSN/1933 virus challenge , but less effective against the rH5N1 virus challenge with a survival rate of 40% . The hH1Chemically inactivated viral vaccines are widely used in clinics for protecting infectious and malignant diseases. However, viral vaccines have disadvantages, such as the risk of virus reactivation, the cost of maintaining vaccine stability, the induction of autoimmune diseases, and deficient protection in some vaccines Phosphorothioate-modified CpG-DNA (PS-ODN), which are resistant to nuclease activity and can be efficiently delivered into cells Divergent strains of avian and human H5N1 influenza A virus have been reported since 1997. It is important, therefore, for the prompt application, to develop the epitope-based peptide vaccine against pandemic influenza. BALB/c mice immunized with a complex of hH5N1 HA233 epitope and Lipoplex(O) produced the epitope-specific IgG , and thein vitro neutralizing activity and in vivo protection effect suggests that other factor(s) such as innate immunity may be involved in the protection in addition to the specific Abs. Taken together, further studies are needed on the prophylactic effect of the complex of hH5N1 HA233 and Lipoplex(O) on the challenge of multiple influenza A subtypes, H1N1 strains, and various highly pathogenic avian influenza A viruses of H5N1 strains. Such studies will allow fine tuning of a potential vaccine, which in turn should lead to improvement of the vaccine program against pandemic influenza.Based on the ability of virus neutralization, sera from mice immunized with hH5N1 HA233 were partially cross-reactive to the H1N1 virus (A/WSN/1933 virus), and swine origin H1N1 virus (A/Korea/01/09) . The vacin vitro or in immunized individuals in vivo when treated with the epitope-specific monoclonal antibodies. We warrant clarifying this issue in the future works. To understand the mechanism involved in the potent adjuvant activity of Lipoplex(O), we also need to clarify other important issues. Human beings have many different genetic backgrounds of HLA types. Therefore, it has to be determined whether IgG production by a complex of B cell epitope and Lipoplex(O) depends on the MHC type and Th1 differentiation and if a novel mechanism is involved.Furthermore, we suggest that the antibodies that recognize hH5N1 HA233 cannot be produced by immunization with an inactivated rH5N1 virus . This reThere are several reports finding B cell epitopes of the HA2 polypeptide and producing monoclonal antibodies through phage-display library selection, HA protein immunization or long size synthetic polypeptide immunization , Table 2In conclusion, we selected the specific B cell epitope hH5N1 HA233, and the vaccine complex of hH5N1 HA233 epitope and Lipoplex(O) stimulated potent protective responses in mice against infection from the rH5N1 virus. To determine the feasibility of developing a vaccine that affords efficient protection against multiple subtypes, we need to ensure that further studies focus on a complex that combines several epitopes together (for example epitopes derived from H1N1 subtypes) with Lipoplex(O).CGTTCGTGTCGGCCTAGCAG. The derivatives and backbone modification of the MB-ODN 4531 sequences and CpG-ODN 1826(S) are described in Limulus amebocyte assay .ODNs were synthesized from Samchully Pharm  and GenoTech . The potent immunostimulatory PO-ODN, MB-ODN 4531 et alet alhttp://tools.immuneepitpoe.org/main/index.html) from the HA protein of the H5N1 A/Viet Nam/1203/2004 strain   . The parAW80717) , Table 5Liposome complexes consisting of peptide and CpG-DNA co-encapsulated in DOPE:CHEMS were prepared as reported previously b) mice were purchased from Central Lab. Animal Inc. . All animal procedures performed in this study are in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research & Quarantine Service of Korea. The protocol was approved by the Institutional Animal Care and Use Committee of Hallym University . The mice were challenged with virus or sacrificed under Zoletil 50+Rompun anesthesia, and all efforts were made to minimize suffering.Mice were maintained under a specific-pathogen-free (SPF) condition. Four-week-old male BALB/c  with 5 µg/ml of peptide and then blocked them with 0.05% of Tween-20 in PBS (PBST) containing 1% BSA. The sera were diluted to 1∶450 with PBST and added to the wells of each plate. The plates were incubated for 2 h at room temperature and washed with PBST. Then, rat anti-mouse biotinylated secondary antibodies  (BD Biosciences) were added to the wells and incubated for 1 h, followed by addition of streptavidin conjugated with horseradish peroxidase for 30 min. To establish the standard curve, purified mouse monoclonal immunoglobulin isotype  standards (BD Pharmingen) were serially diluted in PBST and added to each well. After washing with PBST, biotin-labeled anti-mouse isotype  specific antibody (BD Pharmingen) was added for 1 h. The plates were washed with PBST and incubated with horseradish peroxidase conjugated-streptavidin  for 30 min at room-temperature. A colorimetric assay was developed with a TMB substrate solution , and we used a Spectra Max 250 microplate reader  to measure the absorbance at 450 nm.Madin-Darby Canine Kideny (MDCK) cells were obtained from American Type Culture Collection. The MDCK cells were maintained in Dulbecco’s minimum essential medium supplemented with 10% FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin. Stocks of a recombinant virus, the A/WSN/1933 virus, and the A/Korea/01/09 virus were prepared by inoculation into SPF embryonated chicken eggs or by infection of MDCK cells followed by incubation at 37°C for 72 h. All the experiments involving viruses were performed under a level 2 biosafety condition.Segments of the A/Vietnam/1203/2004 (H5N1) and A/Puerto Rico/8/34 (PR8) (H1N1) influenza viruses were cloned into plasmids for virus rescue by reverse genetics as described previously 3, 1% DEAE Dextran, and (L-tosylamido-2-phenyl) ethyl chloromethyl ketone -treated trypsin. After incubation at 37°C for 3 days, the dishes were stained with 1 ml of crystal violet for 15 min so that we could visualize the plaques. The numbers of plaques were counted to determine the titers.To determine the titers of virus, we performed plaque assays on MDCK cells as described previously 50 for each virus strain, we anesthetized BALB/c mice (N = 6/group) and inoculated them intranasally with serial dilutions of the virus. The survival rates and body weights of the infected mice were monitored every day. The LD50 was defined as the dilution of the virus that produced lethality in 50% of the mice, and LD50 titers were calculated by the method of Reed and Muench To determine the LDVibrio cholerae; Denka Seiken, Japan) overnight and then incubated at 56°C for 30 min to inactivate remaining RDE before use. Approximately 100 PFU/ml of influenza viruses  were incubated with an equal volume of heat-inactivated twofold serially diluted serum samples at 37°C for 1 h. After incubation, the mixtures were added to a confluent monolayer of MDCK cells in a minimum essential medium supplemented with 10% FBS and TPCK-treated trypsin. The cells were cultured for 72 h before the determination of the cytopathic effect. The neutralization percentage was calculated by means of the following equation: Neutralization   =  [/plaque no. of the virus only treated]×100.To confirm antibodies targeting the challenging virus, we performed a virus neutralization assay. The antisera were treated with receptor-destroying enzymes  for 3 days at 37°C in the presence of 1 µg/ml TPCK-trypsin. Plaque assays of the UV-inactivated virus on the MDCK cells demonstrated a complete loss of infectivity.For UV-induced inactivation of the rH5N1 virus (PR8/H5Lo), the virus was irradiated at a distance of 5 cm with 1500 µM/s/cm50 maA/WSN/1933 virus or the 10 LD50 rH5N1 virus. After being infected, the mice were monitored daily for clinical signs and body weight. At the indicated times, the mice were sacrificed by CO2 and all their organs were removed. Organ homogenates were prepared, and the virus titers of each organ were determined by means of a plaque assay. For a histopathologic examination, the lungs were removed and fixed in a 4% buffered formalin solution; they were then embedded in paraffin by a conventional method and cut into 4 µm thick sections. The specimens were stained with hematoxylin and eosin.Four-week-old BALB/c mice (N = 8) were injected i.p. with 50 µg of a complex of peptide and Lipoplex(O) on two occasions at 10 day intervals. Ten days after the second immunization, the mice were intranasally challenged with the 10 LDA/Vietnam/1203/2004 H5N1 HA nucleotide sequence – NC_ AY818135.1.A/Vietnam/1203/2004 H5N1 HA protein – NC_ AAW80717.A/Hong Kong/485/1997 H5N1 HA protein – NC_AAD52043.A/WSN/1933 H1N1 HA protein – NC_AAA43209.A/New York/604/1995 H1N1 HA protein – NC_ ABE11867.A/Wisconsin/4754/1994 H1N1 HA protein – NC_AAB03291.A/Texas/05/2009 A/H1N1 HA protein – NC_ ACP41934.A/Hong Kong/1131/1998 H1N1 HA protein – NC_AAK70451.A/Thailand/271/2005 H1N1 HA protein – NC_ ABK57093.A/Michigan/2/2003 hH1N2 HA protein – NC_ ABI96104.A/Mallard/Alberta/202/96 H2N5 HA protein – NC_ AAT65325.A/Hong Kong/1143/99 H3N2 HA protein – NC_ AAK62039.A/Equine/Jilin/1/1989 H3N8 HA protein – NC_ AAA43151.A/Tern/South Africa/61 H5N3 HA protein – NC_ ABI84970.A/England/268/1996 H7N7 HA protein – NC_ AAC40998.A/Hong Kong/1074/99 H9N2 HA protein – NC_ CAB95857.A/Mallard/Astrakhan/263/1982 H14N5 HA protein – NC_ ABI84453.A/Shelduck/WA/1756/1983 H15N2 HA protein – NC_ ABB90704.A/Duck/Singapore/3/1997 Avian H5 HA protein – NC_ABQ58920.Figure S1Prophylactic efficacy of a complex of hH5N1 HA233 and Lipoplex(O) against influenza A virus. BALB/c mice were immunized i.p. twice with a complex of hH5N1 HA233 encapsulated in indicated combination. The immunized mice were challenged intranasally with the maA/WSN/1933. After the virus challenge, the survival rate (A) and the body weight (B) were recorded for 20 days (N = 8/group). Lipoplex(O), MB-ODN 4531(O) encapsulated in DOPE:CHEMS (1∶1 ratio) complex; LipoplexGC(O), MB-ODN 4531GC(O) encapsulated in DOPE:CHEMS (1∶1 ratio) complex.(TIF)Click here for additional data file.Figure S2Prophylactic efficacy of a complex of hH5N1 HA233 and Lipoplex(O) against influenza A virus. BALB/c mice were immunized i.p. three times with a complex of hH5N1 HA233 encapsulated in indicated combination. The immunized mice were challenged intranasally with the rH5N1 virus (PR8/H5Lo) (A–C). After the virus challenge, the survival rate (A) and the body weight (B) were recorded for 20 days (N = 8/group). The lungs were collected at 3 days, 6 days or 30 days after the challenge with the rH5N1 virus (PR8/H5Lo) (C) (N = 3/group). Scale bars in (C), 100 µm. The lung viral titers were measured by means of a plaque assay to estimate the viral clearance from the lungs at 3 days or 6 days after the challenge with the rH5N1 virus (D). Lipoplex(O), MB-ODN 4531(O) encapsulated in DOPE:CHEMS (1∶1 ratio) complex; LipoplexGC(O), MB-ODN 4531GC(O) encapsulated in DOPE:CHEMS (1∶1 ratio) complex.(TIF)Click here for additional data file.Figure S3Production of cytokines in mice treated with CpG-DNA. BALB/c mice (n = 3/group) were injected i.p with CpG-ODN 1826(S) or Lipoplex(O), and sera from the mice were harvested at the indicated times after injection. The concentration of IL12p40 (A) and IFN-α (B) in the serum was determined by using an ELISA assay.(TIF)Click here for additional data file.


Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway.Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements  at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera  in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production.Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields. Monoterpene biosynthesis and accumulation was indeed shown to localize in the glandular trichomes [An important class of secondary metabolites, terpenoids and isoprenoids contribute more than 50,000 compounds to the rich chemical diversity of natural product structures . Many of (DMAPP) . Two disPP; C20) -9. Monotal parts ,12. Biosrichomes .Saccharomyces cerevisiae is a unicellular eukaryotic organism traditionally regarded as a model system for investigating cellular physiology and widely used as a cell factory for biotechnological applications. Strain improvement has relied in the past on random mutagenesis and classical genetic approaches. In recent years, developments in molecular genetics have made it possible to introduce specific genetic perturbations by modifying the promoter strength of a given gene, to perform gene deletions, or to introduce whole new genes or biosynthetic pathways. Metabolic engineering of an amenable organism such as S. cerevisiae aiming to direct cellular resources and the biosynthetic machinery towards the production of highly desirable natural products, can be an environmentally friendly and economically viable approach to the production of high-added value compounds [S. cerevisiae offers a unique advantage for the production of terpenoids, as the precursors of terpene biosynthesis  are naturally synthesized by the sterol biosynthetic pathway. Recent efforts in yeast metabolic engineering for the production of terpenes have pointed to a significant potential for improvement in the yields of compounds produced [ompounds . S. cereproduced -19.In the current work we aimed to improve yeast strains in a targeted way while avoiding to introduce bacterial plasmid sequences into the yeast genome. The approach was based on recyclable cassette vectors that enable unlimited modifications of the genome. Through the use of the cre-lox recombination system the selection markers can be excised and reused. We employed this approach to introduce strong inducible promoter upstream of endogenous yeast genes and to incorporate at any desired genetic locus extra copies of genes controlled by strong promoters. When applied to the terpenoid biosynthetic pathway, this approach afforded yeast stains which exhibit several fold increase in the yield of monoterpene and sesquiterpene production.S. fruticosa (Greek sage) cineole synthase 1 fused to a hemaglutinin tag (HA), was introduced into EG60 cells and the standard reference laboratory strain BY4741 -CoA into mevalonic acid -ts-hisG-URA3-hisG-HOL DNA fragment -ts, thus generating strain AM65  AM66 (Table Picea abies geranyl diphosphate synthase (GDS) gene was co-expressed. GDS catalyzes the condensation of DMAPP and IPP to GPP, which is the substrate for monoterpene synthases [To improve terpene production by the EG60 strain, we sought to incorporate alterations in the sterol biosynthetic pathway stably into the chromosomal genome avoiding the integration of exogenous bacterial plasmid sequences. As the first step in this approach, the previously developed vector (M4366), containing the employed . The vecemployed ,21. A caradation . The var65 Table . Convers66 Table . To asseynthases . Co-exprERG20 gene was chosen as target for upregulation. ERG20 encodes for a dual specificity enzyme which is primarily responsible for FPP biosynthesis, but also produces GPP  from Salvia pomifera, into the EG60, AM66, and AM68 and AM70 cells generating EG60-03, AM66-02, AM68-02 and AM70-02 respectively . Quantitation of sesquiterpenes was carried out on the registered peak areas of the detected trans-β-caryophyllene, as compared to a standard trans-β-caryophyllene sample . The generated strains were tested for cineole production by expressing SfCinS1 from the pJG4-4 plasmid  led to a significant increase in cineole yield with production reaching 200 mg/L (AM78-01). These levels are 24-fold higher than the standard reference strain BY4741-02 The 3.1 kb cassette was PCR-amplified using primers that would add 50 bp flanking sequences from the d Figure . The IDIL Figure , Table 11 Figure  and 5D. 600) in YPD or galactose-raffinose based medium. The cultures were allowed to grow shaking at 30°C and the OD600 was measured at regular intervals. No inhibitory effect was observed in any of the tested strains in both media. No changes in cell viability were observed 5 days after induction.  or control vectors. This led to the development of the: AM78-03 strain carrying PGal1-IDI1 in pYESmyc and the control PGal1 pYIC1 vector; AM78-04 strain carrying the pYESmyc empty plasmid and the PGal1-IDI1 in pYIC1; AM78-05 carrying PGal1-IDI1 in pYESmyc and PGal1-IDI1 in pYIC1; AM78-06 carrying PGal1-IDI1 in pYESmyc and PGal1-HMG2 (K6R) in pYIC1  variant in AM78-06 cells doubled cineole production  from a plasmid produced 99 mg/g DW, which was 11-fold higher than control cells  it can provide adequately accurate determination of production yields. However, SPME sampling cannot provide information on the levels of compounds retained in the intracellular space, the cell membrane, or in the medium. To assess these quantities and to provide an SPME-independent evaluation of terpene yields in the improved strains, liquid-liquid extractions were performed. Cultures were induced for 3 days and extracted with an 85:15 mix of hexane/ethyl acetate according to the method described by Takahashi . The exts Figure . Extracts Figure .β-pinene reaching 31.59% of the total yield. Limonene and cineole were present in almost equal proportions 13.36% and 12.69% respectively. The SpP330 extraction results gave low yields of only two sesquiterpenes α-humulene at 52.06% and α-guaine at 35.43%, which were only minor compounds by SPME. These differences likely reflect the differential solubility of the products in an aqueous environment or in the cell membrane. Nevertheless, the yields measured using liquid-liquid extractions confirm the positive effect of the applied genetic modifications in terpene production.The relative proportions of terpenes identified by SPME and by liquid-liquid extraction of cineole producing strains differed. The major compound identified was S. fruticosa and S. pomifera in respect with the product profiles identified by SPME GC-MS analysis, of SfCinS1 and SpP330 expressing cells, identified a number of common compounds . All three cDNAs were subsequently identified as multiple isolates. We focused initially on the HSP90 protein, as chaperones are known to affect the folding of their binding partners. Cineole synthase and the HSP90 protein were co-expressed in EG60-06 parental and AM78-09 improved yeast cells. The presence of HSP90 led to a 60% increase in cineole production in EG60-05 cells, and a 20% improvement in the high cineole producing AM78-08 cells , and heme A. Commercial interest for them is very high, for their properties as food colorants and antioxidants (carotenoids) , aroma aHMG1 and HMG2 genes respectively. Between the two HMG genes in yeast, Hmg1p is quite stable, whereas Hmg2p undergoes sterol pathway degradation [HMG2 aimed to maintain the specific localization of the enzyme, which is mostly perinuclear with intense bead-like foci, while suppressing degradation [HMG2 (K6R) either from a high copy number plasmid or from a genome integrated extra copy did not alter the growth properties of the strains used [The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is of key importance in the mevalonate pathway and is subject to feedback regulation as part of the cellular control of sterol biosynthesis . Yeast hradation . Recentlradation . The choradation . Proper radation . Expressins used .HMG2, thus making HMG2 more stable  and AM66 (diploid) which produced substantially higher levels of monoterpenes than parental EG60 cells. Since the COD1 cassette, which was based on D. Stillman's vector [ERG20 gene which encodes the farnesyl diphosphate synthase, a key enzyme catalyzing the sequential head-to-tail condensations of isopentenyl diphosphate  with dimethylallyl diphosphate  and GPP to give FPP [ERG20 allele led to an increase in cineole, as well as sesquiterpene , production . Interestingly, decreasing the abundance of ERG9 squalene synthase by eliminating one allele caused a decrease in monoterpene production, but a substantial increase in sesquiterpene production. This may be due to FPP accumulation which probably inhibits erg20p through product inhibition, thus lowering the availability of GPP [The first cassette developed in this work, COD1, incorporated in the HO locus the K6R variant form of e Figure . The URAs vector , could ogive FPP . Introdu.thionine . Previouthionine ,47. As alability . No farnIDI1, initially from a plasmid and subsequently by integrating a cassette PGal1-IDI1-ts into the mutant leu2 locus of the AM65 strain. The strain developed was capable of high monoterpene production while maintaining robustness, thus enabling more changes in the metabolic pathway to further increase production levels. Using the cre-loxP recyclable cassette system no bacterial plasmid sequences are incorporated in the yeast genome, which would not be desirable for certain applications such as production of compounds as food supplements i.e. carotenoids where consumer acceptance is an issueShifting the balance towards GPP production was achieved by overexpression of Salvia fruticosa, resulted in switching of the product profile in favor of sabinene as sole product [Abies grandis γ-humulene synthase led to the formation of seven enzymes with different spectra of product specificities [The central function of a terpenoid synthase is to bind to the flexible isoprenoid substrate and chaperone the orientation and rotational alterations of the substrate and the reaction intermediates. A significant factor contributing to the multitude of terpenoids found in nature is the formation of multiple products. Almost half of all characterized monoterpene and sesquiterpene synthases also form significant amounts of additional products when the expressed protein is assayed in-vitro . This ma product . This isficities . One parSalvia fruticosa, the three identified proteins are directly relevant to such putative functions. Isolate C4 is a luminal binding protein (BiP), a conserved member of the HSP70 family that is known to bind to incompletely assembled or misfolded proteins in the lumen of the ER [We postulated that an additional strategy to increase production of terpenoids in yeast is by co-expressing plant interacting proteins to biosynthetic enzymes. These interactors could facilitate folding of the enzymes, storage and/or secretion of produced compounds. In the pilot screen performed, using a library from glandular trichomes of f the ER ,51. Lossf the ER . In planf the ER . Isolatef the ER ,55. The IDI1 and HMG2 (K6R) in the AM78 strain yielded 60% higher monoterpene levels than co-expression in the parental background, indicating that the system is not yet saturated. Additionally, we aimed to stabilize the plant cineole synthase SfCinS1, by co-expressing its interacting partner HSP90, which also led to production increases.Yeast strains exhibit high variability in terpenoid production. A suitable wild type strain was selected for improvement. A series of cassettes were developed which allow the integration of any desired genetic element into the yeast genome at suitable loci, avoiding the introduction of unnecessary exogenous DNA sequences. The tools were applied for the generation of new strains with upregulated biosynthesis of terpenoid precursors. The improved yeast strains yielded higher terpenoid levels. Co-expression from high copy number plasmids of SPME fiber 2 cm-50/30um DVB/Carboxen™/PDMS StableFlex™ Fiber (Supelco);. 1,8 cineole , γ-terpinene , α-pinene , β-myrcene (M-0382) and (-)-trans-caryophyllene , α-copaene  and a 70% Sabinene solution kindly donated by VIORYL Chemicals, were used as standard compounds. Myc-Tag (9B11) mouse mAb, ; HA-Tag (6E2) mouse mAb,  and Anti-Mouse IgG (Fab specific)-Peroxidase antibody  were used for protein detection. Yeast media: D (+)-Glucose monohydrate ; D-(+) Galactose ; Raffinose pentahydrate ; Yeast Nitrogen Base w/o AA, carbohydrate & w/AS ; Complete Minimal (CM) medium is composed of 0.13% (w/v) dropout powder , 0.67% (w/v) yeast nitrogen base w/o AA, 2% glucose. For galactose based medium glucose is substituted with 2% galactose, 1% raffinose; 5'-Fluoroorotic acid monohydrate ; β-glucuronidase (G7017 Sigma); TOPO TA Cloning Kit Dual Promoter ; SuperSignal West Pico Chemiluminescent Substrate .URA3, 2μ, Gal prom.); pYES2-myc ; pJG4-4; pJG4-6 ; pJG4-5 ; pYX143 ; pYX143-HA ; pYES-ADH1 . The pYIC1 vector was constructed by integration of the LEU2 orf, which was PCR amplified using 5'URA-LEU 5'-ACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAA TCATGTCGCCCCTAAGAAGATCGT-3' and 3'URA-LEU 5'-AGTTTAGTATACAT GCATTTACTTATAATACAGTTTTTTAAGCAAGGATTTTCTTAACTTC -3' primers, into the pYES2 plasmid replacing the URA3 marker. The success of recombination was confirmed by PCR and auxotrophic complementation of the new vector. The gene PCR amplifications were performed using Platinum Taq (Invitrogen) or Hotstar Taq (Qiagen) were cloned by TOPO TA cloning into the pCRII vector (Invitrogen). The cloned amplified products were submitted for DNA sequencing prior to additional manipulations. The open reading frame minus the transit peptide (orf) of S. fruticosa cineole synthase 1  was PCR amplified from a previously isolated full length cDNA [Picea abies) isoprenyl diphosphate synthase PalIDS1 (Acc. GQ369788) was PCR amplified from a full length cDNA, kindly donated by Dr. Gershenzon using the primers 5'GPPS(EcoRI) 5'-GAATTCATGTGCTCAAACACAAATGCCCAG-3' and 3'-GPPS(XhoI) 5'-CTCGAGTCAGTTCTGTCTTTGTGCAATGTA-3'. The S. pomifera putative sesquiterpene synthase P330 was PCR amplified from a previously isolated full length cDNA clone (unpublished data) using the primers 5'P330(BamHI) 5'-TGGATCC GAGCTGAAATATATGCATCGGCT-3' and 3'P330 5'-TGATGGAGTTTGATTC TAGACCT-3'. The Arabidopsis thaliana HSP90 orf was PCR amplified from the recombinant clone RALF06-10-K09, (RIKEN Arabidopsis) using the following primers: 5'HSP90(BamHI) 5'-GGATCCATGGCGGACGCAGAAACCTT-3' and 3'HSP90(XhoI) 5'-CTCGAGGTCAACTTCCTCCATCTTGCT -3'. Expression of the proteins was verified in pYX143-HA, pJG4-6, pYES-myc constructs in western blots using antibodies against the hemaglutinin tag or the myc tag. However, in most cases untagged vector counterparts were used to eliminate the possibility of any interference of the tags.The following expression plasmids were used for expression in yeast cells: pYES2  5'-ATGTCACTTCCCTTAAGAAC GATAGTACATTTG-3' which introduced a point mutation at K6 converting it to R and a 3' primer 3'HMG2(XhoI) 5'-CTCGAGTTATAATAATGC TGAGGTTTTACAGGGGGG-3'. The PCR product was cloned by TOPO TA cloning (Invitrogen) and sequenced. The galactose promoter -multicloning site(mcs)-terminator sequence (ts) was PCR amplified from a yeast expression vector using primers 5'GAL(MfeI) 5'-CAATTGTTTAAACGGATTAGAAGCCGCCGAGCG-3' and 3'CYC1(BglII) 5'-AGATCTGGCCGCAAATTAAAGCCTTCG-3'. The fragment was cloned by TOPO TA cloning and was subsequently transferred into the M4366 vector (Acc. AF324729) generating the construct M4366-Gal1p . The HMG2 (K6R) cDNA was excised with EcoRI and the insert was ligated into the M4366-Gal1p vector at the EcoRI site. The orientation of the cDNA relative to the promoter was first assayed using primers from the PGal1 and the 3' of the gene -ts-hisG-URA3-hisG-HOL) was linearized by digestion with NotI and was transformed into the EG60 strain. Colonies growing in Glucose/CM-ura were selected and tested for integration using genomic DNA as template and primers from the PGal1 and the HMG2 (K6R) cDNA. The strain was named AM63. The URA3 selection marker was excised by plating cells on 5-FOA plates which counter-select for the presence of URA3. The hisG sequences flanking URA3 recombine causing the elimination of the gene. Growing colonies were tested by replica plating on Glucose/CM-ura and were found to be ura deficient. A strain of the opposite mating type containing the same COD1 cassette was crossed to AM63 to create a diploid strain homozygous for COD1. The strain was named AM66.Using genomic DNA from the e Figure . The posSfiI site at 5' of the PGal1 promoter. The amplified product was cloned into the pCRII TOPO vector. The construct which contained the SfiI site of the PGal1 proximal to the HindIII site of the vector was used for additional manipulation. The loxP-URA3-loxP fragment from the pUG72 vector was excised by digestion with HindIII and SfiI and inserted into the pCRII- PGal1 plasmid, giving rise to COD2 cassette . The ADH1 promoter was PCR amplified using the primers SpeI-SfiI 5'ADH 5'-ACTAGTGGCCTATGCGGCCTATTTCGGATATCCTTTTGTTG-3' and 3'- ADH (HindIII) 5'-AAGCTTGGAGTTGATTGTATGCTTG-3' which introduce and SfiI and a HindIII site at the 5' and 3' of PAdh1 respectively. The PCR product was cloned and inserted into the pUG72 vector as above, giving rise to the COD3 cassette (loxP-URA3-loxP-PAdh1.)The galactose promoter was PCR amplified using the primers 5'-GAL(SfiI) 5'-AGTGGCCTATGCGGCCACGGATTAGAAGCCGCCGA-3' and 3'-GAL 5'-AATTCGGATCCAGAGGCATAATCTGGCACATCATACATG-3' which introduce an Gal1 upstream of the ERG20, using as a template the COD2 cassette the loxP-URA3-loxP- PGal1-HA sequence was PCR amplified by using the following primers: ERG20-COD2-for 5'-CTCAACCAACAGGTATTGGACTGACATAGGCACAATAAACTCAAA AATAAAGCTTCGTACGCTGCAGG-3' and ERG20-COD2-rev 5'-GGGAAAACGTTC AAGAATCTCTCTCTCCTAATTTCTTTTTCTGAAGCCATGGATCCAGAGGCATAAT CT-3'. The products of six PCR amplifications were pooled and purified by phenol/chloroform extraction and ethanol precipitation. The DNA was used in a lithium acetate transformation to transform AM66 cells. Colonies growing in glucose/CM-ura plates were selected and tested for proper integration of the promoter using genomic DNA from the URA+ colonies and primers 5'GALprom and 3' ERG20 confREV 5'-TTGGAAGGTGACCTCATG GAACAATTCG-3'. The PCR products were resolved by agarose gel electrophoresis. Six out of eight tested colonies carried the specific integration. The strain was named AM67. Excision of the URA cassette was achieved by transformation with the plasmid pB227Galp-Cre which expresses the Cre recombinase under the control of the galactose promoter. Cells harbouring the plasmid were plated in Galactose-raffinose/CM-leu media and grown colonies were tested for uracil auxotrophy. The selected ura cells were cured of the plasmid and were named AM68.To integrate PGal1-mcs-ts was PCR amplified from an expression vector using the primers 5'-GAL (HindIII) 5'-AAGCTTACGGATT AGAAGCCGCCGAG-3' and 3'-ts 5'-GTCGACGGCCGCAAATTAAAG CCTTCG-3'. The amplified PCR product was purified by gel extraction and cloned into the pCRII vector in a TOPO TA reaction. The pUG27 vector [IDI1 orf was inserted into the COD4 plasmid using the EcoRI and XhoI restriction sites found at the mcs generating the COD40 cassette. The previously mutated leu2 gene was chosen as the site of integration of the COD4 cassette. The cassette was amplified in PCR reactions using the primers LEU2-pUGF 5'-ATGTCTGCCCCTAAGAAGATCGTCGTTTTGCCAGGT GACCACGTTGGTCAGCTTACGGATTAGAAGCCGC-3' and LEU2-pUG Rev 5'-AG CAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGATAGGC CACTAGTGGATCTG-3' and purified as above. The DNA was used to transform AM65 cells. Transformed cells were selected for growth in glucose/CM- his plates. The cells were named AM77.The Phis5+ from S. pombe flanked by loxP sites was PCR amplified using primers EGR9-pUG F 5'-AGAGAAAAGACGAAGAGCAGAAGCGGAAAACGTATACACGTCACATA TCACAGCTGACTTCGTACGC-3' and ERG9-pUG R 5'-GTACTTAGTTATTGT TCGGAGTTGTTTGTTTATGTTATTTGGCGCAGACTGCATAGGCCACTAGTGGATCTG-3'. AM68 cells were transformed as above and colonies growing in glucose/CM-his. The stable integration of disruption cassette was confirmed by PCR amplification of a 500 bp fragment using the primers ERG9prom 5'-CTAAA CGAGCAGCGAGAACACGACCAC-3' and pUG27confR 5'-GGATGTGATGT GAGAACTGTATCC-3'. The PCR product was resolved by electrophoresis on 1% agarose gel.To generate a deletion in one of the two alleles of the diploid AM68 cells, the pUG27 cassette containing the S. cerevisiae strains carrying the plasmids for terpene expression were initially grown in 5 ml overnight cultures in glucose based selective media at 30°C. In the following day 50 ml fresh glucose media was inoculated from the grown cells to an OD600 = 0.1 and incubated shaking at 30°C until the OD600 reached 0.5-0.7. The cultures were pelleted and washed twice with sterile ddH2O. The washed pellets were resuspended in 50 ml of Galactose-Raffinose based selective growth medium and incubated at 30°C ~12 hours. The induced cultures were either kept in the medium and assayed in regular intervals, or washed twice with sterile ddH2O and resuspended in 20 ml of buffer  and incubated at 30°C from 12 hours to several days. When the incubation on buffer was prolonged more than 12 hours the accumulated volatiles were released every 24 hours. Measurements to assess levels of production were performed on cells kept in galactose-raffinose media, while product identification assays and assays for extended periods were performed on cells kept in buffer. Volatiles sampling was performed using a Solid Phase Microextraction (SPME) assembly. The SPME method was performed by using 2 cm-50/30um DVB/Carboxen™/PDMS StableFlex™ Fiber for Manual Sampling. Yeast cells, resuspended in 20 ml of buffer  and incubated at 30°C, were sampled by exposing the SPME fiber for 30 min into the headspace (using a 250 ml Erlenmeyer flask). 1,8 cineole, γ-terpinene, α-pinene, β-myrcene and (-)-trans-caryophyllene were used as standard compounds. They were dissolved in pentane and ten-fold serial dilutions were prepared. 10 μl from each dilution was added to 20 ml buffer solution and exposed to SPME sampling in identical conditions to the cultures tested, and subsequently measured by GC. The GC cineole peak area values were plotted against the concentration of the compound.The developed The exposed SPME fiber was withdrawn into the outer septum-piercing needle, removed from the flask, and inserted in the heated injection port of the gas chromatograph. The products were identified by comparing retention times and mass spectra with authentic reference compounds. In-vitro enzyme assays were performed in yeast cell extracts broken by sonication according to the method described by Kampranis et al . The GC 2 steam to approximately 10% of the original volume and then applied to a silica gel column (5 × 70 mm). Terpenes were then eluted with 90% pentane, 10% diethyl ether in serial fractions. The presence of the products in the separated fractions was confirmed by thin layer chromatography and the identified fractions carrying the products were further analyzed by GC-MS. The amount of purified product was reported to the dry weight (DW) of the corresponding cell pellet.Liquid-liquid extraction was performed with selected yeast strains which were evaluated for terpene production using 100 ml cultures incubated at 30°C for 3 days. The cultures were extracted with 15% ethyl acetate, 85% hexane . The celThe SfCinS1 PCR amplified insert cloned into the pCRII TOPO vector was digested with EcoRI and XhoI and subcloned into the pGILDA vector linearized with the above restriction enzymes. The composite construct fuses in frame the LexA moiety of the vector with CinS1. Expression of the LexA-CinS1 was verified using antibodies against the LexA part of the fusion protein. A tomato cDNA library cloned into the pJG4-5 vector was screened as previously described ,56. InteSPME: Solid Phase Micro Extraction; CM medium: Complete Minimal medium; FOA: syn. 5'-FOA, 5'-Fluoroorotic acid; HMGR: syn. HMG-CoA reductase, 3-hydroxy-3-methyl-glutaryl-CoA reductase; MVA: Mevalonate pathway; DMAPP: Dimethylallyl pyrophosphate; syn. Dimethylallyl diphosphate; IPP: (3-isopentenyl pyrophosphate; syn. 3-isopentenyl diphosphate; GPP: geranyl pyrophosphate; syn. GDP: geranyl diphosphate; FPP: farnesyl pyrophosphate; syn. FDP: farnesyl diphosphate; GGPP: Geranyl geranyl pyrophosphate; syn. GGDP: Geranyl geranyl diphosphate; ER: Endoplasmic reticulum.The authors declare that they have no competing interests.CI, IC, SL and PK conducted the experimental work. CBJ and SCK assisted in the data analysis and review of the manuscript. AMM conducted some experiments and is responsible for the design and drafting the paper. All authors have read and approved the manuscript.


Saccharomyces cerevisiae, posing the question how most yeast promoters nucleate PIC assembly. Here we show that one third of all yeast promoters contain a novel conserved DNA element, the GA element (GAE), that generally does not co-occur with the TATA box. The distance of the GAE to the transcription start site (TSS) resembles the distance of the TATA box to the TSS. The TATA-less TMT1 core promoter contains a GAE, recruits TBP, and supports formation of a TBP-TFIIB-DNA-complex. Mutation of the promoter region surrounding the GAE abolishes transcription in vivo and in vitro. A 32-nucleotide promoter region containing the GAE can functionally substitute for the TATA box in a TATA-containing promoter. This identifies the GAE as a conserved promoter element in TATA-less promoters.Initiation of RNA polymerase (Pol) II transcription requires assembly of the pre-initiation complex (PIC) at the promoter. In the classical view, PIC assembly starts with binding of the TATA box-binding protein (TBP) to the TATA box. However, a TATA box occurs in only 15% of promoters in the yeast  In eukaryotes, transcription of protein-coding genes relies on RNA polymerase (Pol) II, the general transcription factors (GTFs) TFIIB, -D, -E, -F, and –H, and coactivators such as Mediator and the Spt-Ada-Gcn5 acetyltransferase complex (SAGA) Since TATA-less promoters require TBP for function in vivo reporter gene assays, and in vitro biochemistry to identify and functionally characterize a region in TATA-less yeast core promoters that is bound by TBP, required for Pol II transcription and contains a novel conserved promoter element, the GA element, or GAE.Based on these observations, we hypothesized that TATA-less core promoters contain DNA elements that are functionally similar to the TATA box in promoting PIC assembly and Pol II transcription. Here we used a combination of bioinformatics, In contrast to higher eukaryotes, where the distance between the TATA box and the TSS is fixed at around 30 nucleotides, a variable distance of 40−120 nucleotides is observed in yeast, apparently due to a TSS scanning mechanism Sc, S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus). We allowed for a shift of up to +/− 3 nucleotides between motifs in the multiple sequence alignments. Again, the GAE stands out as being the most highly conserved of all the more frequent 5-mers . This search identified a DNA element comprising one guanine followed by four adenines (GAAAA) as being most highly anti-correlated to the TATA box , only trSc promoters containing only a GAE are three times more abundant than promoters containing only a TATA box  stretches, which are proposed to occlude nucleosomes from promoters TMT1 promoter function is strongly dependent on the sequence context in the GAE-containing region.Activity of the adenines , lane 10adenines  in vitroHIS4 and TMT1 promoter DNA. TBP bound HIS4 DNA as expected, but also bound the TATA-less TMT1 DNA  and fluorescently labeled DNA encompassing 40 base pairs of TMT1 DNA . As expein vivo and in vitro. We found that the HIS4 TATA box can functionally replace the 12-nucleotide GAE-containing region in the TMT1 promoter  tract in the ILV1 promoter decreased transcription significantly without affecting nucleosome organization in vivoSeveral studies of poly(dA:dT) tracts in individual model promoters indicated a role in promoter function that is linked to nucleosome positioning Sc, S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus) were used. For calculating the number of mutations, the start position in a window of +/−3 bp with the least number of mutations to the motif in Sc in every species was used.The TSS used for our analysis were generated by merging the cDNA data set of Miura et. al HIS4 (428 bp upstream to 24 bp downstream of the start codon) and TMT1 (273 bp upstream to 24 bp downstream of the start codon) and their mutant variants were cloned between HindIII/BamHI into a pRS315 plasmid with the lacZ gene inserted between NotI/SalI. Reporter plasmids were transformed into BY4741 wild type yeast cells and grown in biological duplicates in SD (–leu) medium to an OD600 of 0.5–1.0. β-galactosidase levels were determined using the Yeast β-galactosidase Assay Kit .The native promoter sequences of www.fhcrc.org/science/basic/labs/hahn) 33]. In vitro transcription and analysis by primer extension were performed as described 33]. Primer extension for all constructs was done using the same 5’-Cy5-labeled oligonucleotide (5’-TTCACCAGTGAGACGGGCAAC). For activated transcription 200 ng of recombinant full-length Gcn4 was added. Template plasmids were generated by inserting the respective promoter sequence as described above in pBluescript KS+ with HindIII and BamHI. The Sc Gcn4 gene was inserted into vector pET21a with NcoI and SacI introducing an N-terminal hexahistidine tag. Transformed E. coli BL21 (DE3) RIL cells (Stratagene) were grown in LB medium at 37°C to an OD600 of 0.5. Expression was induced with 0.5 mM IPTG for 16 h at 18°C. For protein purification, cells were lysed by sonication in buffer A . After centrifugation, the supernatant was loaded twice onto a 2 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed three times with 10 column volumes (CV) of buffer A containing 20 mM imidazole. Bound protein was eluted with 10 CV of buffer A containing 500 mM imidazole. Protein was further purified by cation exchange chromatography using a HiTrap SP column  equilibrated with buffer B . The protein was eluted with a linear gradient of 10 CVs from 0 to 1 M NaCl in buffer B. After concentration, the sample was applied to a Superdex 200 size exclusion column (Amersham) equilibrated with buffer C . The sample was concentrated to approximately 0.5 mg/ml, flash frozen in small aliquots in liquid nitrogen, and stored at −80°C. All buffers contained a protease inhibitor cocktail.Nuclear extracts were prepared from 3 l of BY4741 wild type yeast culture as described  in a total reaction volume of 20 µl. Recombinant TBPcore and TFIIB were expressed in E. coli and purified as described 35]. Proteins were diluted in dilution buffer . Samples were incubated for 30 min at 18°C and loaded on a 6% native polyacrylamide gel . The gel was pre-run for five min and then run at 160V for 15 min at room temperature in 190 mM glycine, 25 mM Tris pH 8.3 23°C. Gels were analyzed with a typhoon scanner FLA9400 and ImageQuant Software . The oligos used for the assay had the following sequence (only coding strand is shown):Electrophoretic mobility shift assays (EMSA) were performed as described 34], with minor modifications. Templates were generated by annealing the complimentary PAGE-purified oligonucleotides (coding strand was 5’-Cy5-labeled). The binding reaction contained 3 nM of 40 bp 5’-Cy-5-labeled dsDNA, 300 nM TBPHIS4 (TATA), 5’-ACAGTAGTATACTGTGTATATAATAGATATGGAACGTTAT;HIS4 (GC-8), 5’-ACAGTAGTATACTGTGGGCCGGCCAGATATGGAACGTTAT;HIS4 (GAE), 5’-TAGTATACTGTGAAAGGAAAAGGAAGATATGGAACGTTAT;TMT1 (GAE), 5’-ATTTTCATTTTTAAAGGAAAAGGAATGTAACTAATTTAGT;TMT1 (GC-12), 5’-ATTTTCATTTTTGGCCGGCCGGCCATGTAACTAATTTAGT;TMT1 (TATA), 5’-ATTTTCATTTTTTATATAATATGTAACTAATTTAGT;ATTTTCATTTTTAAAAAAAAAAAAATGTAACTAATTTAGTTMT1 (A-12), 5’- Figure S1The GAE can occur in multiple copies. Fraction of sequences with the TATA box (purple) and GAE (green) in at least a given number of multiple occurrences per sequences in 3711 Sc promoters aligned at the TSS.(EPS)Click here for additional data file.Figure S2Frequency of the GAE with different length of A-tracts in 3711 Sc core promoters.(EPS)Click here for additional data file.Figure S3Only double stranded templates are bound by TBP and TFIIB. Electrophoretic mobility shift assay. A 5’-Cy5-labeled 40 bp single (ssDNA) and double stranded (dsDNA) templates were incubated with recombinant TBPcore alone or TBP core and TFIIB. The protein-DNA complex was separated from free DNA by native polyacrylamide gel electrophoresis. Free DNA and bound protein-DNA complexes are indicated.(EPS)Click here for additional data file.Figure S4GAE function depends on promoter context. (A) and (B) Electrophoretic mobility shift assay. A 5’-Cy5-labeled 40 bp double stranded DNA of the respective promoter construct was incubated with recombinant TBPcore alone or TBP core and TFIIB. The protein-DNA complex was separated from free DNA by native polyacrylamide gel electrophoresis. Free DNA and bound protein-DNA complexes are indicated. (C) Promoter activity in an in vivo β-galactosidase reporter gene assay and in vitro transcription (txn) assay of the wild type and mutated Sc SER3 promoter. The 8-nucleotide TATA box of the SER3 promoter was substituted by the 12-nucleotide GAE-containing region of the TMT1 promoter.(EPS)Click here for additional data file.Figure S5The GAE-containing region can functionally replace the TATA box. (A) Schematic depiction of promoter substitution constructs of the TATA-less TMT1 and the TATA-containing HIS4 promoter. TATA box and bioinformatically defined GAE are marked by a black box. Solid and dashed lines represent HIS4 and TMT1 promoter sequences, respectively. TSS of HIS4 and TMT1 are labeled with a and b, respectively. Half-arrows mark primer annealing sites for primer extension reaction. (B) Promoter activity in an in vivo β-galactosidase reporter gene assay and in vitro transcription (txn) assay of the wild type and mutated HIS4 and TMT1 promoter. Negative control (lane 8) is background signal of the reporter plasmid without a promoter. Specific transcripts from the HIS4 TSS and TMT1 TSS are marked by a and b, respectively.(EPS)Click here for additional data file.Figure S6GAE has no inhibitory effect on the TATA-containing HIS4 promoter. Promoter activity in an in vivo β-galactosidase reporter gene assay of the wild type and mutated HIS4 promoter. Promoter substitution constructs as indicated on the right. Negative control is background signal of the reporter plasmid without a promoter.(EPS)Click here for additional data file.Figure S7The GAE anti-correlates with SAGA-dominated genes. Percentage of Sc core promoters containing TATA box, GAE, both elements or none of the elements in SAGA- and TFIID dominated Sc promoter classes (EPS)Click here for additional data file.Table S1All Sc promoters containing a bioinformatically defined GAE.(XLS)Click here for additional data file.


Multiple subtypes of avian influenza viruses have crossed the species barrier to infect humans and have the potential to cause a pandemic. Therefore, new influenza vaccines to prevent the co-existence of multiple subtypes within a host and cross-species transmission of influenza are urgently needed.Here we report a multi-epitope DNA vaccine targeted towards multiple subtypes of the influenza virus. The protective hemagglutinin (HA) antigens from H5/H7/H9 subtypes were screened for MHC II class-restricted epitopes overlapping with predicted B cell epitopes. We then constructed a DNA plasmid vaccine, pV-H3-EHA-H1, based on HA antigens from human influenza H3/H1 subtypes combined with the H5/H7/H9 subtype Th/B epitope box.P < 0.05). The multi-epitope group significantly enhanced Th2 cell responses as determined by cytokine assays. The survival rate of mice given the multi-epitope vaccine was the highest among the vaccine groups, but it was not significantly different compared to those given single antigen expressing pV-H1HA1 vaccine and dual antigen expressing pV-H3-H1 vaccine (P > 0.05). No measurable virus titers were detected in the lungs of the multi-epitope immunized group. The unique multi-epitope DNA vaccine enhanced virus-specific antibody and cellular immunity as well as conferred complete protection against lethal challenge with A/New Caledonia/20/99 (H1N1) influenza strain in mice.Epitope-specific IFN-γ ELISpot responses were significantly higher in the multi-epitope DNA group than in other vaccine and control groups (This approach may be a promising strategy for developing a universal influenza vaccine to prevent multiple subtypes of influenza virus and to induce long-term protective immune against cross-species transmission. Over the years, influenza has become a serious public health problem. With the potential for sudden outbreaks, rapid spread, and high incidence of complications, the prevalence of influenza infections has caused tremendous loss of human life and material resources ,2. Thus,Based on their internal protein antigens, different influenza viruses can be divided into 3 types: A, B, or C. The surface antigens, hemagglutinin (HA) and neuraminidase (NA) are also used to identify different subtypes. At present, the prevalent human influenza viruses are the type A H3/H1 and type B viruses. However, in recent years, multiple subtypes H5/H7/H9) of the avian influenza virus (AIV) have been able to cross the species barrier to infect humans . Around /H7/H9 ofImmunogenic epitopes in an antigen is determined by the major histocompatibility complex (MHC) class I for cytotoxic T cell lymhocytes (CTL) and MHC class II for T helper (Th) cells. These polymorphic MHC molecules present short peptides that are processed after an exogenous antigen  is taken up by antigen presenting cells (APC) such as macrophages and dendritic cells. These APC then "present" the peptide to the immune cells that recognize the MHC/peptide complex via the T cell receptor (TCR) or B cell receptor (BCR). Theoretically, given any set of MHC II restricted peptides presented to the Th cells, the optimal sequence would be those that could also stimulate B cells to produce antibodies since activation of antigen-specific Th cells also promote antibody production. By understanding the specific epitopes from pathogens that can stimulate optimal immune responses, we will better understand how to tailor vaccines to a specific population and/or pathogen.Indeed, many studies have shown the efficacy of peptide-based vaccines in animal models , as wellPresently, standard inactivated vaccines based on one or a few circulating strains are mainly utilized for prevention of influenza infection, but they cannot effectively deal with the current trend of increasing variations of the circulating viruses. A new influenza vaccine that can afford long-term and cross-species protection against multiple subtypes of influenza is imperative. Developing DNA vaccines that can stimulate both humoral and cellular immunity is a promising area of research. In particular, a multi-epitope DNA vaccine which expresses antigen genes in tandem can efficiently present the defined protective epitopes to stimulate the immune system while eliminating non-essential components or potential toxic fragments of traditional inactivated vaccines. Additionally, the development of such multivalent vaccines can be combined with other vaccine antigens to enhance immunogenicity. The advantage of combination vaccines is that they can potentially provide broader coverage to protect against rapidly mutating viruses such as influenza.We report here the generation and evaluation of the immunogenicity of a DNA vaccine expressing HA based on human influenza H3/H1 combined with a class II MHC multi-epitope antigen (hereafter referred to as the "multi-epitope" vaccine). The vaccine was evaluated for induction of humoral and cellular immune responses in a mice model as well as for the protective efficacy against lethal H1N1 subtype virus challenge. We expected the vaccine targeted towards human influenza subtype H3 and H1 to provide total protection against these strains while at the same time achieving some level of protective efficacy against other influenza subtypes. This approach may be effective against rapidly mutating influenza and provide longer-term protection while laying the foundation for development of a new universal influenza vaccine.Female BALB/c mice (6-8 weeks old) were used for immunization and challenge studies. All mice were maintained with free access to sterile food and water.CY033622) and A/Wisconsin/67/2005 (H3N2) strains were stored in the laboratory. Virus stocks were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs for 48 h at 37°C. The viruses were titrated by the Reed and Muench method to determine the median lethal dose (LD50). Baby hamster kidney (BHK-21) cells were used for transient expression experiments. All experiments with influenza viruses were conducted under BSL-3 containment, including work in animals.A/New Caledonia/20/99 influenza virus (H1N1) ), AAR02636 [A/Netherlands/127/03(H7N7)], and DQ997437 [A/swine/Shandong/nc/2005(H9N2)]. MHC II restricted epitopes were predicted bioinformatically by the network server SYFPEITHI and Multipre, and B cell epitopes were predicted using the network server BCEPRED http://www.imtech.res.in/raghava/bcepred/ or the Biomolecule simulation software Insight II . Th cell epitope predictions were based upon their cumulative binding affinity to six of the most common HLA-DRß1 alleles . The network server BCEPRED was used for linear B cell epitope prediction which screens sequences based on hydrophilicity, accessibility, flexibility, antigenicity, polarity, and exposed surface residues. The Th epitope prediction was narrowed down to include as much as possible the epitopes which overlapped with the predicted B cell epitopes in order to obtain epitopes with dual functions of stimulating both T and B cells.The HA gene sequences of the influenza H5, H7, H9 subtypes which have crossed species barriers to infect mammals and become vaccine strains were downloaded from NCBI The Th/B cell epitope box was designed with "GPGPG" linkers between each epitope in order to reduce interference between epitopes and to ensure the proper processing and function of each epitope independently. The "KK" linker was also added to prevent the epitopes between subtypes from "splitting", that is, to avoid generation of new junctional epitopes ,17. BaseNhe I/Hind III, Cla I, Xho I, Cla I/Xho I and Hind III/Xho I, respectively, to yield pMD18-H3HA, pMD18-H1HA1 and pMD18-EHA. A eukaryotic expression vector, pVAX1  was used to construct the following DNA vaccine vectors: pV-H3HA, pV-H1HA1, pV-H3-H1 and pV-H3-EHA-H1. The four DNA constructs were sequenced to confirm cloning accuracy before amplification in Escherichia coli JM109 and purification using endotoxin-free kits . The final DNA preparations were resuspended in sterile saline solution and stored at -20°C until further use.The HA genes of influenza A/New Caledonia/20/99 [H1N1] and A/Wisconsin/67/2005 [H3N2] were obtained by RT-PCR amplification of the isolated RNA. The H3HA, H1HA1 and the epitope box (termed EHA) sequences were inserted into the pMD18-T vector after addition of restriction sites BHK-21 cells were transfected with purified DNA from pV-H3HA, pV-H1HA1, pV-H3-H1, pV-H3-EHA-H1 and pVAX1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. In brief, cell monolayers were grown on glass coverslips in a 6-well plate and were then transfected with the plasmid DNA (10 μg/well). At 48 h after transfection, the cells were fixed with 0.05% glutaraldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS), followed by incubation with rabbit anti-HA of A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/2005 (H3N2), A/Indonesia/5/05(H5N1), A/Netherlands/127/03(H7N7), A/swine/Shandong/nc/2005(H9N2) polyclonal antibody [1:200 in poly (PBST)] for 1 h at 37°C. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibodies [in PBS/bovine serum albumin (BSA)] were added and then incubated for 1 h at room temperature. After mounting the samples, fluorescence images were scanned using an Olympus microscope .50  of the A/New Caledonia/20/99 H1N1 virus in a final volume of 100 μL. The challenge experiments were performed in a biosafety level 3 (BSL3) facility .In challenge experiments, four DNA vaccine groups and an empty vector pVAX1 control group of female Balb/c mice (n = 15) were immunized intramuscularly . The immunization schedule consisted of 2 administrations with a 3-week interval, and bleeding was performed at 0, 1, 2, 3, 4 and 5 weeks after immunization for determination of antibody titers. To assess the efficacy of the cross-protective immunity of the 2 vaccine doses against lethal challenge 2 weeks after the second immunization, the immunized mice were anesthetized and intranasally challenged with 10 LDTo determine tissue viral titers, the lungs of surviving mice challenged with H1N1 were collected and homogenized by mechanical disruption. The viral titers were determined by plaque formation assay performed in MDCK cells in the presence of trypsin as previously described ,19.A pre-coated enzyme-linked immunosorbent assay (ELISA) kit was used  to determine the cytokines levels of interferon (IFN)-γ and interleukin (IL)-4 in the immunized the mice according to the manufacturer's instructions. Serum samples (100 μl) from different groups of mice were tested in duplicate. After 36 h of incubation with the standards and samples, the plates were washed, followed by addition of 50 μl of the Streptavidin-HRP solution. The plates were incubated at 37°C for 60 min before washing again for at least five times, with a 1-2 min interval in between each wash. The diluted substrate was added at 50 μl per well and incubated at 37°C for 15 min. Finally, 50 μl of stop solution were added per well to terminate the reaction. Absorbance values were measured at 450 nm. Standard curves were drawn according to the instructions of the kits. The cytokines levels in the samples were calculated accordingly, expressed as ΔX ± SD, and differences between groups were analyzed statistically.6 cells/well) was prepared and stimulated with 20 μg/ml of the inactivated whole virus antigen preparations of A/New Caledonia/20/99 (H1N1) and A/Wisconsin/67/2005 (H3N2) or the following HA antigen peptides (20 μg/ml) of A/Indonesia/5/05(H5N1), A/Netherlands/127/03(H7N7), and A/swine/Shandong/nc/2005(H9N2): H5HA141-155, H5HA206-223, H5HA302-316, H7HA165-181, H7HA255-269, H7HA182-196, H9HA123-140, H9HA73-90, H9HA37-54. The plates were placed in a CO2 incubator at 37°C. The following day, the splenocytes were discarded, and the plates were extensively washed with pre-chilled PBS. IFN-γ spots were detected by a biotinylated anti-mouse IFN-γ specific antibody, followed by addition of streptavidin-horseradish peroxidase (HRP) and development with 3-amino-9-ethylcarbazole (AEC) substrate solution. The spots were counted using an automated ELISpot reader. The results were expressed as the number of spot-forming cells (SFC)/106 spleen cells. P-values were calculated using a permutation test stratified for the experiment.The frequencies of IFN-γ secreting splenocytes were analyzed using a commercially available mouse IFN-γ pre-coated ELISpot assay according to the instructions of the manufacturer . Lymphocytes from the spleen were removed aseptically 10 days after a boost immunization, and a single cell suspension  or standard antigens of H5, H7 and H9 subtype were coated overnight in 96-well plates . Following blocking of non-specific binding, the serum samples were diluted 100 times in PBS containing 0.5% (wt/vol) gelatin, 0.15% Tween 20, and 4% calf serum (ELISA diluent) and applied in duplicate wells for a 1 h incubation at 37°C. The plates were washed five times with PBS and then reacted with a 1:2000 dilution of HRP-labeled goat anti-mouse IgG (Zhongshan Goldenbridge Biotech) for 1 h at 37°C. After another five washes with PBS, the substrate was added (10 mg ortho-phenylenediamine [OPD] + 20 mL 0.015% hydrogen peroxide in phosphate/citrate buffer). After incubation for 15 min at 37°C, the reactions were terminated with 2N HP-values of 5% or less were considered statistically significant. The data was analyzed using the SPSS Version 16.0 software.The Lifetest procedure using the Kaplan-Meyer method and log rank test were applied for survival analyses between study groups . All tests applied were two-tailed, and SYFPEITHI and Multipre were used in different algorithms to predict Th epitopes. BCEPRED is an improved linear B cell epitope prediction method that utilizes multi-parameter analysis to predict potential B cell epitopes. Comprehensive analyses of both Th and B cell epitopes were performed to obtain a set of epitopes in which the predicted Th epitopes would also contain potential B cell epitopes. The selected epitope regions were re-evaluated for their spatial conformation and specificity to determine the final epitopes Figure . A finalBefore testing the immunogenicity of the vaccines, the four DNA vaccine constructs Figure  were conP < 0.01) than that of the control group, indicating that all of the vaccines tested effectively stimulated Th1 type responses. The multi-epitope group displayed the highest level of IFN-γ secretion, although the difference was not significant compared with the other 3 groups (P > 0.05).Fourteen days after the boost immunization, the sera were collected and analyzed for IFN-γ and IL-4 levels levels by ELISA Figure . The ordP < 0.01) than that of the pVAX1 control group. The IL-4 levels in the serum of the multi-epitopes group were significantly higher than that of the other immune groups (P < 0.05), indicating that the immunized groups had significantly enhanced Th2 cell function. Combined with the analysis of IFN-γ levels above, these findings demonstrated that the multi-epitope vaccine induced the greatest levels of vaccine specific immune responses in mice, and the use of these epitopes tended to produce Th2 cytokines and promoted humoral immunity.As for the detection of IL-4 levels in the immune sera, the order was determined as follows: multi-epitopes immune group  > two-subtype co-expression immune group  > H3 immune group  > H1 immune group  > pVAX1 control group (188). The IL-4 levels of the immune groups were also significantly higher (in vitro incubation with whole inactivated viruses [influenza virus A/Wisconsin/67/2005 (H3N2) and influenza virus A/New Caledonia/20/99 (H1N1)]  compared to other immunized groups. However, the multi-epitope DNA group did have significantly (P < 0.05) higher levels of IFN-γ secretion than the other groups in response to peptide antigens , but the levels of the different vaccine groups were not significantly different from each other (P > 0.05). That is, the IgG antibody levels induced were equivalent between the multi-epitope vaccine group (pVAX1-H3-EHA-H1), the single antigen expressing groups (pV-H3HA and pV-H1HA1) and the dual antigen expressing group (pV-H3-H1).In evaluating the development of virus-specific IgG against the H3 and H1 subtypes of influenza by ELISA Figure , the antP < 0.01) at 35 DPI. Furthermore, the Th/B multi-epitope group (pV-H3-EHA-H1) had significantly higher H5, H7 and H9 subtype IgG levels than the other immunized groups , suggesting that the selected epitopes could effectively stimulate virus-specific antibodies. The highest antibody levels detected by ELISA were against the H5 epitopes, suggesting that this vaccine would theoretically be more effective against this virus subtype in mice.From the analysis of H5, H7, H9 subtypes of influenza virus-specific IgG antibodies Figure , the var50 of A/New Caledonia/20/99 (H1N1) influenza strain. Their survival rates were monitored for the following 14 days. The mice began to show clinical signs or death from influenza infection on day 5 post-challenge. Figure P < 0.01) than that of the pV-H3HA and pVAX1 control groups  were immunized IM with 200 μg of each vaccine and challenged with 10 LD weeks weThe lungs were harvested from mice which survived the viral challenge, and viral titers were determined by plaque formation assays in MDCK cells. Because almost all the mice which received the pVAX1 control and pV-H3HA immunizations did not survive, lungs from dead mice in these groups had to be selected to test for viral titers. As expected, the mice of the pVAX1 control group and pV-H3HA group all had positive viral titers in the lung. By contrast, no measurable virus titers were detected in the lungs in the multi-epitope immunized group, and somewhat lower levels of virus  were observed in the pV-H3-H1 and pV-H1HA1 immunized groups Figure .In light of the recent 2009 H1N1 pandemic, there is an urgent need to develop new influenza vaccines. New influenza vaccines should have the following characteristics: low cost, high level immunogenicity, rapid preparation, protection against rapid virus mutation and long-term protection against multiple subtypes of influenza, especially against potential influenza pandemic strains.The purpose of this study was to further develop and evaluate a novel approach to vaccination based on multi-subtype influenza epitopes using mice as a mammalian model. We assessed the immunogenicity of an H3/H1-derived multi-epitope DNA vaccine and its protective efficacy against H1N1 virus challenge. The experiment was set up to systematically compare the specially designed multi-epitope vaccine with the separate antigen components of the immunized groups and a control group.The MHC II molecule pathway of antigen processing is first activated in an APC by phagocytosis, pinocytosis, or receptor-mediated endocytosis of an exogenous antigen. The phagocytic lysosome products are digested into linear epitopes and then later associated with MHC II molecules to be presented on the surface of the APC. Th cells and APC recognition between cells and signal transduction and the resulting induced Th cell activation play an important role in the initiation of an acquired immune response, maintenance of responses in chronic infections and development of immune memory. Activated Th cells produce cytokines that can effectively regulate cytotoxic T cells, B cells and phagocytic cell functions .B cell epitopes form the basis of humoral immunity in that they determine the specificity of antibodies. B cells can capture antigens through the BCR and function as APC to activate Th cells. Activated Th cells can also activate B cells in turn to produce antibodies against the corresponding antigen. An ideal immunogenic epitope is one that elicits responses from both Th and B cells . TherefoP > 0.05). However, the stimulation of IL-4 levels by the multi-epitope vaccine was significantly higher than the other vaccine groups (P < 0.05), indicating that the multi-epitope pV-H3-EHA-H1 vaccine could enhance Th2 cell immune function. Therefore, the multi-epitope vaccine tended to induce a dominant Th2 response which could then promote humoral immune responses.IFN-γ is the defining Th1 type cytokine, with important immunoregulatory functions including the ability to activate macrophages, induce monocyte cytokine secretion, affect the body's Th1/Th2 balance, regulate antigen presenting cells, and significantly increase MHI-1 and MHC-II molecule expression . IL-4 isP < 0.05), which confirmed that epitopes in a multi-epitope vaccine could induce functional antibodies, especially those of the H5 subtype.Production of antibodies against viral infection is an important effector function, and the level of humoral immunity reflects the ability and strength of the body to block infection, to rid itself of the virus if infection occurs, or at least to prevent tissue damage . In persP > 0.05). When stimulated with peptides associated with the H5, H7, H9 antigens, the splenocytes of the multi-epitope vaccine group produced the highest number of IFN-γ spots which was significantly different from that of the other groups. Together these results indicate that the HA subtype and the specific epitopes may play major roles in inducing Th1 cellular immune responses.The DNA vaccines also effectively stimulated cellular immune responses in mice as evaluated by the IFN-γ ELISpot assays. For the H3 and H1 IFN-γ responses, the multi-epitope group produced the highest number of spots, but not significantly different compared with the single antigen expressing and the dual antigen expressing groups . No measurable virus titers were detected in the lungs of the mice in the multi-epitope immunized group. Somewhat lower virus titers were observed in the pV-H3-H1 and pV-H1HA1 groups, which indicated that the constituent epitopes contributed to the cross-protective immunity against H1N1 viral challenge.Further evidence of the efficacy of the multi-epitope vaccine was provided by the Overall, the multi-epitope DNA vaccine induced significant levels of humoral and cellular responses as well as provided cross-protective immunity. This study demonstrates the proof-of-principle that a universal DNA vaccine with engineered epitopes may protect against multiple subtypes of influenza virus, afford long-term immune protection, and prevent cross-species transmission.The authors declare that they have no competing interests.LT performed most of the experimental work and drafted the manuscript. DZ, BH and ZYW participated in the analysis of humoral and cellular responses. MYT participated in the immunization of mice. NYJ and HJL revised the manuscript for important intellectual content and gave final approval of the version to be published. All authors read and approved the final manuscript.