Team:Slovenia/Notebook/Methods

PROTEIN PRODUCTION, ISOLATION AND MODIFICATION

The complete coding regions of H. pylori flaA from the H. pylori SS1 strain and fliC from Escherichia coli K12 were amplified by PCR and cloned into AK3 or pET19b vector (Novagen). Chimeric flagellins were constructed via PCR ligation and Biobrick assembly methods. For the DNA cloning experiments E.coli  DH5α strain was used. After DNA sequences verification E.coli BL21(DE3)pLysS were transformed with specific constructs for the overexpression of flagellar proteins. Cultures were grown in LB media (Luria-Bertani) supplemented with ampicillin at 37°C with shaking at 180 rpm until the OD600 was 0.4-0.5. At this point temperature was shifted to 25°C for maximal yield of native protein in supernatant. To induce protein expression 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added when OD600  was 0.7-1. Bacterial cells were further cultivated overnight and then harvested by centrifugation (5000g, 10 min at 4°C). Bacteria were resuspended in lysis buffer (0.1% sodium deoxicholate, 10 mM Tris/HCl pH 8.0) with protease inhibitors (CPI, Sigma) and sonicated for homogenisation. After centrifugation, supernatant with soluble fraction of His-tagged recombinant flagellin was loaded on a pre-conditioned Ni-NTA column (Ni-NTA Agarose, Qiagen), which was then washed in 50 mM Tris/HCl, 100 mM NaCl, 20 mM imidazole, pH 8.0). Proteins were eluted with buffers, containing 50 mM Tris/HCl, 100 mM NaCl, and imidazole with concentrations of 50 mM and 250 mM. Fractions that spectrophotometrically indicated typical protein peaks at 280 nm were combined and concentrated. Purity and integrity of proteins was assessed by SDS-PAGE and by Western blot. Proteins were separated on 10% SDS-PAGE gels and stained with Coomassie Blue or transferred for 60 min at 350 mA to nitrocellulose membranes (Amersham). Membranes were blocked with 0.2% I-Block (Tropix) in PBS-T (1x PBS/0,1% Tween-20) for 60 min, then incubated sequentially with primary mouse anti-His monoclonal antibody (Qiagen) for 90 min at a dilution of 1:1000 and peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) for 45 min at a dilution of 1:3000 at room temperature. The signal was detected with chemiluminescence substrate (Pierce). As proteins were determined, we dialyzed them two times against 1x PBS buffer. Secondary structure was examined by CD spectroscopy.

CIRCULAR DICHROISM

Circular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to help determine the structure of macromolecules (including the secondary structure of proteins). This method was used to confirm that intact protein with it's proper secondary structure was isolated. CD pectra were  taken the far-UV region between 190 and 250 nm on a Chirascan CD spectrometer (Applied Photophysics). The cell path length used was 1 mm with sample concentration 0.5 mg/ml in MQ water.

FLUORESCENT PROTEIN LABELING

Proteins were fluorescently labelled with Alexa Fluor 555 hydrazide (Molecular Probes), incubated with 100 mM natrium  borate  (pH 8.5) for 2 h at room temperature with shaking (protected from light). Afterwards, 10-fold excess of Tris buffer (pH=7) was added to deactivate remainder of Alexa dye, after it  has reacted with primary amines.  Finally, to remove unbound Alexa Fluor 555,  fluorescently labeled proteins were extensively dialyzed against PBS buffer. 

ELISA

ELISA (Enzyme-Linked ImmunoSorbent Assay) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In our experiments ELISA was used to determine mouse serum IgG antibody response to the recombinant protein vaccination. 96-well microtiter plates were coated either with 50 µl of each recombinant protein antigen (conc. 10 µg/ml) or heat killed H. pylori preparation from 50 million cells in the volume of 50 µl overnight at 4°C. After blocking  with 3% BSA in PBST (150 mM NaCl, 7.5 mM Na₂HPO₄, 2.5 mM NaH₂PO₄, 0.05% Tween-20) overnight in a humid atmosphere at 4°C and washing three times with PBST, appropriate dilutions of sera were added to the wells, and incubated for 90 min at 37°C to enable specific IgG antibody binding to immobilized antigens. Following a washing step, horse radish peroxidase-conjugated secondary antibodies with a specifity for the mouse IgG class of antibodies were added at a dilution of 1:3000. After another 90 min incubation at 37°C plates were washed again to remove unbound secondary antibodies. Finally, ABTS (Sigma) substrate was added and after 20 min incubation the reaction was stopped with 1% SDS. Absorbance values were quantified at 450 nm in a multilabel plate reader Mithras (Berthold Technologies).

FLOW CYTOMETRY

Flow cytometry employs instrumentation measuring the fluorescence on individual cells. Cells are suspended in individual droplets, squirted past a laser that excites any fluorescent dye in them and past a detector that measures the fluorescence. Fluorescence measurement was carried out on a Epics Altra (Beckman-Coulter Electronics) Flow Cytometer Cell Sorter.

REACTIVITY OF SERUM ANTIBODIES WITH H. pylori

In our experiment we used fluorescently labeled antibody fragment goat F(ab)2 anti-mouse IgG-PE to detect IgG reaction with H. pylori. Collected H. pylori cells were washed with FACS (10x PBS, 0.5% BSA, 0.05% Azide) buffer and incubated  for 30 min on ice  in 100 µl FACS buffer containing  8 µl serum. After that bacteria were washed again and incubated for 30 min on ice in FACS buffer containing F(ab)2 goat anti-mouse IgG fragment, conjugated to phycoerythrin (Beckman Coulter), to a final concentration 0.01 mg/ml. After another washing and resuspension step, samples were placed in reaction tubes and analyzed with flow cytometry. 

FLOW CYTOMETRY PROTOCOL FOR INTRACELLULAR STAINING OF HEK293T

48h prior intracellular staining HEK293T cells were transfected with 2000 ng of indicated construct using GeneJuice Transfection Reagent (Novagen). We began intracellular staining with resuspension of cells in PBS + 3% FBS. Then we fixed the cells 10 min at room temperature by adding 4% paraformaldehyde. Cells were washed two-times with PBS + 3% FBS. Next, we permeabilized cells with 0.5% Tween-20 30 min at 4°C. Cells were then stained with primary antibodies (mouse anti-HA antibodies, Invivogen, final concentration 0.016 mg/ml) 40 min at 4°C and then with secondary antibodies (goat anti-mouse IgG-PE, Beck.Coul., final concentration 0.01 mg/ml) for 30 min at 4°C. After that we washed cells two-times and analyzed them on flow cytometer.  

FLOW CYTOMETRY PROTOCOL FOR SURFACE STAINING OF HEK293T 

48h prior intracellular staining HEK293T cells were transfected with 2000 ng of indicated construct using GeneJuice Transfection Reagent (Novagen). We began surface staining with resuspension of cells in PBS + 3% FBS. Then we fixed the cells 10 min at room temperature by adding 4% paraformaldehyde. Cells were washed two-times with PBS + 3% FBS. Cells were then stained with primary antibodies (mouse anti-HA antibodies, Invivogen, final concentration 0.08 mg/ml) 40 min at 4°C and then with secondary antibodies (goat anti-mouse IgG-PE, Beck.Coul., final concentration 0.01 mg/ml) for 30 min at 4°C. After that we washed cells two-times and analyzed them on flow cytometer.

CYTOKINE TESTING

For detecting production of cytokines upon cell activation with DNA TLR vaccines we used real-time PCR and an Multiplex Fluorescent Bead Immunoassay (Bender MedSystems) for quantitative detection of human cytokines by flow cytometry.

HEK293 cells were seeded into a 12-well plate and transfected with the indicated DNA TLR vaccines or wtTLR plasmid controls. 24 h post transfection the DNA TLR vaccine transfected cells were lysed for RNA extraction and positive control (wtTLR-vector transfected) cells were stimulated for 4 h with the corresponding ligands (LPS and poly(I:C)) and then lysed for RNA extraction. Supernatants from the same cells were collected for the Flurescent Bead Immunoassay.

MULTIPLEX FLUORESCENT BEAD IMMUNOASSAY

Supernatants were collected from cells as described above and analysed using the FlowCytomix Human Basic Kit by Bender following manufacturer's instructions and flow cytometry. Briefly, beads of different sizes and spectra addresses are coated with antibodies reacting with each of the cytokines being detected in the samples. The two different bead sizes make in possible to detect up to 20 analytes in a single small volume. Next a biotin-conjugated secondary antibody is added to the bead-antibody-cytokine complex, and binding is detected with streptavidin-phycoerythrin with flow cytomety.

REAL-TIME PCR

Real-Time or quantitative PCR has become a popular technique with which to obtain insight into the complexity of the immune response. The easy detection of cytokine mRNA transcripts in a limited number of cells where the corresponding protein could barely be measured is the major advantage of the technique. Real-time PCR is so called because the amplicon accumulation can be directly monitored during the PCR process, using fluorogenic probes or fluorescent dyes that intercalate with dsDNA.

Cells were prepared as described above; harvested, lysed and total RNA was isolated with Trizol reagent according to manufacturer instruction (Gibco Life Technologies). All solutions were treated with the RNase inhibitor DEPC (Sigma). DNAse was used to remove genomic DNA from RNA samples and reverse transcribed with the QuantiTect Reverse Transcription Kit (Qiagen). The real-time PCR was performed with the QuantiTect SYBR Green PCR Kit (Qiagen) on the Roche LC 480. We used primers, specific for IFN-β, IFN-α, IL-1α, IL-6 and IL-8. Data was analyzed with the LightCycler 480 Software.

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CONFOCAL MICROSCOPY

Confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional  images by using a pinhole to reject out of focus fluorescent light in specimens that are thicker than the focal pane. In is widely used in life sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labeled with one or more synthetic fluorescent probes, immunoflourescent reagents or contain fluorescent proteins.

SUBCELLULAR LOCALISATION OF DNA TOLL-LIKE RECEPTORS VACCINES

For surface staining HEK293T cells were seeded into Ibidi 8-well μ-slides and 24 h after that transfected with 250 ng of indicated constructs using GeneJuice Transfection Reagent (Novagen). 48 h post transfection they were fixed for 10 min at room temperature by adding 4% paraformaldehyde and after that blocked with 3 % BSA in PBS. Next, cells were stained with primary antibodies (mouse anti-HA antibodies, Invivogen, final concentration 0.016 mg/ml in PBS with 3% BSA) for 60 min at 37°C, washed 3 times with PBS and then stained with secondary antibodies in the dark (goat anti-mouse IgG-FITC, Imgenex, final concentration 1.5 ng/ml in PBS with 3% BSA) for 45 min at 37°C. After that cells were washed 4 times with PBS and imaged using a Leica TCS SP6 confocal microscope with a 488 nm laser beam.

UPTAKE OF FLUORESCENT LABELED FLAGELLIN

Cells were seeded into 8-well μ-slides and after 24 h transfected with TLR5 receptor or kept untransfected for blank control. Proteins were directly administered to HEK293 cells 24 h post-transfection. As a negative control, Alexa Fluor 555 was diluted in 1x PBS buffer with its absorbance matching that of the chimeric protein. Simultaneously, Alexa Fluor 633-labeled human transferrin (Invitrogen - Molecular Probes) was added to label early endosomes. The samples were incubated for 1h at 37°C, washed with PBS and imaged using a Leica TCS SP6 confocal microscope with a 543 nm and a 633 nm laser beam.

SURFACE EXPRESSION OF CHIMERIC FLAGELLIN

To evaluate whether chimeric proteins are expressed on the bacterial surface, bacteria were transformed with several chimeric flagellin-coding plasmids and grown in liquid meida. 5 h after IPTG induction they were harvested and incubated with mouse anti-His antibodies (Qiagen) for 1h at room temperature. After that they were washed 3 times with PBS buffer and incubated for another 1h with goat anti-mouse IgG-FITC (Imgenex) at room temperature in the dark. Stained bacteria were visualized using a Leica TCS SP6 confocal microscope. 

BACTERIAL MODIFICATION

Recombinant flagellin composed of N- and C-termini of E. coli flagellin and the variable domain of H. pylori flagellin, was fused with H. pylori antigens. The constructs were cloned downstream of the IPTG-inducible T7 promotor. Some constructs were also cloned under constitutive TetR promotor control. As carrier strain we used a non-motile fliC mutant of E. coli, strain JW1908-1 (CGSC #9586). As the mutant has no gene for the T7 RNA polymerase, we used the λDE3 Lysogenization Kit (Novagen) to integrate λDE3 prophage into the E. coli host cell chromosome. λDE3 is a recombinant phage carrying a cloned gene for T7 RNA polymerase under lacUV5 control.

We also used the bacterial ghost (BG) system as a novel vaccine delivery vehicle (kindly provided by Haas). BG are nonliving Gram-negative empty bacterial envelopes. They enhance immune responses against envelope-bound antigens, including mucosal immunity and T-cell activation. Further advantages of bacterial ghost vaccines are safety, simplicity of the production method and the fact that they can be stored and processed without the need for refrigeration. E. coli strain JW1908-1 were transformed with a BG plasmid, coding the lysis gene E under a temperature controlled promotor in order to lyse and empty the bacteria of their cytoplasmic contents. Controlled expression was induced at 42° C.

PROTEIN EXPRESSION IN MODIFIED BACTERIA

To show expression under control of the T7 promotor, we transformed E. coli JW1908-1 with a GFP construct in the pET19b vector. Bacteria were grown in LB medium. The addition of IPTG induced the expression of GFP, assessed by flourimetric measurements. The same experiment was used to prove GFP expression under control of the constitutively active tetR promotor.

LUCIFERASE REPORTER ASSAY

The dual luciferase reporter assay was used to evaluate the activation of TLR5 with flagellin and constructs that comprise citosolic signaling (TIR) domain of TLR3, TLR4 or TLR9.

For signalization studies reporter genes are used, that are composed of a promotor, which is activated upon TLR activation of interest and a gene coding for light-emitting enzymes. In our experiments firefly luciferase (fLuc) gene under control of NFκB or IFNβ inducible promotor was used as a reporter for TLR5, 4 and 9 or TLR3 activation, respectively. Apart from fLuc reporter and TLR constructs, HEK293 cells were cotransfected with another luciferase reporter (coding for Renilla luciferase) under constitutive CMV promotor control. Its activation-independent expression and specificity for a different substrate than fLuc enabled us to normalize fLuc activity measurements and transfection efficiency validation.

EXPERIMENTS WITH ISOLATED CHIMERIC PROTEIN

The main form of bacterial flagellin in vivo is polymerized protofilament, flagellar filament. To be recognized by TLR5, flagellin must undergo depolymerization. It might occur during phagocytosis of activated cell or during bacterial growth and replication (Smith et al, 2003). Thus, flagellin filaments used for assessing biological activity in some experiments were depolymerized by heating for 20 min at 70°C. In experiments with isolated monomeric chimeric flagellin depolymerization was not needed.  HEK293 cells were seeded into a 96-well clear-bottom plate (Corning-Costar) and after 24 h transfected with the following plasmids: 20 ng wtTLR5, 50 ng fLuc, 10 ng rLUC per well and JetPEI transfection reagent (Polyplus). 24 h post transfection they were stimulated with recombinant Salmonella typhimurium flagellin (for positive control) and isolated chimeric flagellin for 6 h and lysed. Luciferase activity was measured using multilabel reader Mithras.

EXPERIMENTS WITH MODIFIED BACTERIA AS MUCOSAL VACCINES

Bacteria were grown in LB medium with required antibiotics (kanamycin, chloramphenicol and ampicilline). Protein expression was induced with IPTG. After the cell density was high enough (0,8 to 1,0 at 600 nm), bacteria were washed with sterile 1x PBS to remove growth medium and IPTG.  After  washing they were diluted 1:2000. One bacterial sample was lysed by cooking at 70° C for 20 minutes, while the other one was stored at 4° C. Both samples were tested on HEK293 cells for TRL5 activation with the dual-luciferase reporter assay. 

EXPERIMENTS WITH DNA FLAGELLIN VACCINE

Cells were co-transfected with TLR5 and several constructs that activated TLR5 signaling pathway and both luciferase reporter plasmids. Such cells produced TLR5 and at the same time recombinant flagellins with a signal sequence for transport outside the cell. By doing so, they were able to activate themselves. Secreted recombinant flagellins activated the TLR signaling pathway through binding to TLR5, located at the cell membrane.

TLR5 activation in mixed cell lines. We separately transfected HEK293 cells with the TLR5 plasmid and luciferase coding plasmids as described above and with DNA constructs coding for chimeric flagellin. 24 h post transfection cells were mixed in a 1:1  ratio and left to reattach to the plate. After another 24 hours cells were lysed and luciferase activity was measured. 

EXPERIMENTS WITH DNA TOLL-LIKE RECEPTORS VACCINE

Cells were transfected with DNA constructs that comprise the TIR domain of the TLR3, TLR4 or TLR9 and an agent for dimerization. 30 or 40 hours (for TLR3 or TLR4 and 9 constructs, respectively) after transfection cells were lysed and luciferase activity was measured to test their constitutive signaling activity.

EXPERIMENTS PERFORMED ON LABORATORY MICE

STRAIN OF MICE AND HOUSING

The mouse strain C57BL/6J was obtained from the breeding centre of the Medical faculty, University of Ljubljana, Slovenia. This is a well characterized strain (genome and physiology) and was shown to be responsive to H. pylori infection by exhibiting higher colonization rates and increased patho-histological changes in the stomach upon infection. In addition, this strain reacts to the infection mostly by Th1 cell response, which resembles the response of humans to infection with H. pylori. We can therefore better extrapolate the results gained in this mouse model to the human. All procedures were approved by the Veterinary Administration of the Republic Slovenia and the Ethical committee for the laboratory animals following all the current Slovenian legislation that has been harmonized with the EU legislation on the use of laboratory animals in research. Female animals (5 per cage or treatment) were housed at the age of 8-10 weeks in the facility of the Institute for Microbiology (Medical Faculty, University of Ljubljana). Animals were individually numbered by ear-notching, regularly examined, weighed and fed a standard rodent chow (Altromin 1324, Lage, Germany). Food was available ad libitum, except on the night before oro-gastric applications took place. The mice had access to water at all times.

VACCINATION WITH THE PROTEIN VACCINES

Two engineered flagellin protein vaccines were tested, CF-UreB (chimeric flagellin  with Urease B) and CF-multi  (chimeric flagellin with multiepitope). Lysozyme (Sigma) was used as the negative control. These proteins dissolved in PBS were mixed with adjuvant aluminum hydroxide (Imject Alum, PIERCE) in a ratio of  2 (protein) to 1 (AlOH). The final amount of the protein applied intraperitoneally was 100 ug in a volume of 300 ul. Booster re-vaccination was again performed intraperitoneally 10 days later.

VACCINATION WITH BACTERIA, THAT PRODUCE H. pylori ANTIGENS

Bacteria producing chimeric flagellin with multiepitope in the middle (CF213-multiepitope-CF215) were applied either by oro-gastric application (directly to the stomach) in a volume of  100ul (2.2 X 10⁸ CFU per animal), while another group received intranasal application of 2.2 X 10⁷ CFU in the final volume of 10 ul of sterile 0.9% saline solution. Mice receiving bacteria with the backbone plasmid only served as negative controls.

VACCINATION WITH DNA VACCINES

DNA vaccines were introduced by electroporation (Tevz et al., 2008) into the leg muscles or subcutaneously. Constructs tested were:

a)CMV-HA-UreaseB-His6;

b)CMV-ss-HA-UreaseB-His6;

c)CMV-ssCF-UreB (BBa_K133018)

d)CMV-CF-multi-CF;

e)CMV-SS-HA-multiepitope-TMTIR4-His6;

f)CMV-SS-HA-multiepitope-CD4(ecto+TM)-TIR3-His6;

g)CMV-SS-HA-ureaseB-CD4(ecto)-TMTIR9-His6

Electroporation or electropermeabilisation is a significant increase in the electrical conductivity and permeability of cell plasma membrane caused by externally applied electrical field. It is usually used in molecular biology as a way of introducing specific substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or, as in our case, a piece of coding DNA. Pores in the cell wall are produced when the voltage across the membrane exceeds its dielectric strength. If the strength of the applied electrical field and/or duration of exposure to it are properly chosen, there will be no permanent damage to the cell - the pores will close upon a period of time, while the molecules will have enough time to enter the cell. If a cell is exposed too often or too strongly to electrical field, it can result in apoptosis or necrosis.

DNA constructs were isolated using Mobius 1000 Ultra plasmid isolation kit (Novagen), diluted in sterile PBS at the final concentration of 1 mg/ml. All constructs were applied subcutaneously (50 µl) using a  thin needle 29G (Myector, Terumo Japan). Some constructs (c, e) were also applied intramuscularly to the right leg  muscle (musculus tibialis cranialis), to which 20 µl of the DNA solution was injected. Mice were anaesthetized by isoflourane- nitrous oxide and oxygen inhalation. First, the hair was removed from the area of application. Two parallel electrodes made of stainless steel (30mmx10mm, 6mm distance, IGEA, Carpi, Italy) were placed to the area of DNA vaccine injections and electric pulses were triggered using the Cliniporator (IGEA, Carpi, Italy). Subcutaneous electroporation was performed using a pulse of  600 V/cm, 100 ms followed by a pulse of  84 V/cm 400 ms, 1 Hz. Between the pulses there was 500 ms pause. Intramuscular electroporation was performed by a pulse of 360 V/cm, 100 ms followed by 4 pulses of  48 V/cm, 100 ms, 1 Hz. The mice were boostered 10 days after the first vaccination using the same procedure described above.

BLOOD SAMPLING

Each mouse was placed in a special ventilated and heated cage to enable peripheral vasodilatation for faster and easier blood sampling. The animal was then fixed in a restrainer and local anesthetic ethyl chloride was applied to the end of the tail. About 80-100 ml of blood was collected from the tail end and the tail tip was sealed with silver nitrate to stop bleeding.