Yeast strain and growth conditions
P. pastoris GS115 (Invitrogen, USA) was grown at 28°C in YPD medium (Yeast Extract Peptone Dextrose Medium). For growth on plates, 2% agar was added to the media. Transformants were grown in media supplemented with 250-750 μg/ml Geneticin (Sigma, USA). For cloning procedures, Escherichia coli DH5 α were used and grown at 37°C in LB medium supplemented with 50 μg/ml either kanamycin or ampicillin.
Molecular biology protocols were carried out according to Sambrook and Russell . E. coli and P. pastoris cells were transformed by Electroporation. Enzymes Eco RI, Not I, Sal I and T4 DNA ligase (Fermentas, USA), Taq DNA polymerase and reagents for PCR (Invitrogen, USA) were used as recommended by the supplier. DNA sequencing was performed by ABI DNA sequencer (Applied Biosystems, USA). DNA sequences were analyzed using DNA star software.
Cloning H1N1 HA gene into pPICK9K yeast transfer vector
The DNA corresponding to nucleic acids 1 to 1699 of the HA gene from novel California/04/2009 H1N1 [Genbank:FJ966082.1] was synthesized by Biotech desk (Hyderabad, India). The full length HA-encoding synthetic gene was PCR amplified from the synthetic construct using high fidelity Pfu Taq polymerase (Fermentas, USA) employing the following primers having introduced Eco RI and Not I sites in the forward and reverse primers respectively. H1N1HA Forward: 5'- TTG GAT CCA GAA TTC ATG AAG GCA ATA CTA GTA GTT CTG-3' [Base pairs: +1 to +24 Genbank: FJ966082.1]; H1N1HA reverse: 5'-TGG ATC CGC GGC CGC AAT ACA TAT TCT ACA CTG TAG AGA -3' (Base pairs: +1699 to +1681 NCBI Genbank: FJ966082.1]. The PCR conditions used were: 94°C for 45 sec, 63°C for 45 sec, 72°C for 1 min and 30 sec, for 35 cycles, and finally 72°C for 10 min. The amplified HA gene was digested with Eco RI and Not I restriction enzymes and was cloned into pPIC9K yeast transfer vector (Invitrogen, USA) at the same restriction sites. The resulting vector pPICK9KH1N1HA (Figure 1) had the HA gene in frame with the fused Saccharomyces cerevisiae α-mating factor secretion signal under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. [The complete sequence of the resultant pPIK9KH1N1HA recombinant construct is submitted to NCBI Genbank: HQ398363.1]. The pPICK9KH1N1HA DNA was transformed into E.coli DH5 alpha strain (Invitrogen, USA) via heat shock method. For selection of the recombinant transformants, the bacterial cells were cultured in Luria-Bertani medium (Himedia, India) supplemented with 50μg/ml ampicillin and 50 ug/ml of Kanamycin. The positive bacterial transformants were selected through restriction digestion of plasmid DNA using Eco RI and Not I enzymes and PCR analysis using alpha factor forward (5'- TAC TAT TGC CAG CAT TGC TGC-3') and H1N1 HA reverse primers. Correct integration will result in the formation of a 1.96 Kbp PCR product. The complete HA gene sequence was further confirmed through nucleotide sequencing using ABI sequencer for any possible mutations introduced during PCR step.
Integration of pPICK9KH1N1HA DNA into Pichia pastoris genome and screening of the transformants
The recombinant plasmid DNA pPIC9KH1N1HA was linearized by digesting with Sal I enzyme to integrate the transgene at His4 locus on the Pichia genome and also to generate HIS+, Mut+ transformants in Pichia pastoris GS115 cells. Ten microgram of the linier DNA was used to transform fresh electro competent P. pastoris cells via electroporation using Bio-Rad Gene Pulsar Xcell™ electroporation system (Bio-Rad laboratories, Inc USA.) at three different voltages (1600V, 1800V and 2000V), 20μF capacitance and 200Ω resistance. After transformation, cells were plated on SD-His plates (1.34% yeast nitrogen base, 2% dextrose, 0.01% complete amino acid supplement minus Histidine, 1 M sorbitol supplement, and 2% agar), and incubated at 30°C for 2 days. The parent pPIC9K without insert, linearized with Sal I was also transformed similarly for negative control. The colonies obtained were streaked on fresh SD-His plates. It is often desirable to select for transformants containing multiple integration events (Figure 1B) as such clones potentially express significantly higher levels of the recombinant protein. Three hundred and sixty five transformed colonies bearing the chromosomally integrated copies of the pPICK9KH1N1HA were screened for single, double or multiple copy integrants through replica plating on YPD plates containing different concentrations of Geneticin (250μg, 500μg and 750μg). Plates were incubated at 30°C for four days and the growth obtained was scored with plus (+) and minus (-) for the presence or absence of growth respectively on the selection plate. Since both KanR and H1N1HA gene are integrated together, resistance to Geneticin would indicate the copy number of the integrated H1N1 HA gene. As a reference, clones that grow well on selection plate with Geneticin concentration of 250μg/ml, 500μg/ml and 750μg/ml were considered to have single, double and more than four copies integrated respectively. To further confirm the transformants having multiple copy integrants, PCR was performed on the genomic DNA isolated from selected colonies by employing alpha factor secretary signal forward and H1N1HA reverse primers. A total of twenty clones were randomly picked up from all the three categories (3, 6 and 11 clones each from 250μg/ml, 500μg/ml and 750μg/ml plates respectively) and were inoculated into 10 ml of YPD broth and grown at 28°C until the OD was 2-3. The OD in all was finally adjusted to 2 using the blank YPD medium and 10 ml of each culture was further used for Genomic DNA extraction. The template DNA concentrations in all cases were equally adjusted in the PCR reactions to check its sensitivity to screen single, double and multiple copy integrants along with appropriate control as reported earlier . Fourteen PCR positive Pichia clones that were found to contain single, double and more than four copies of the H1N1HA gene integrated were selected further for subsequent expression studies.
Expression analysis and optimization of H1N1 HA protein expression in Pichia system
Fourteen PCR positive His+ Mut+ Pichia clones were selected for methanol induction. The glycerol stocks of the above Pichia clones were inoculated separately into 50-ml of either YPGy (1%Yeast extract, 2%bacto peptone and 1%glycerol buffered with 100 mM potassium phosphate buffer, pH 8.2) or Buffered Minimal Glycerol medium-BMGM (100 mM potassium phosphate, pH 8.2, 1.34% YNB, 4 × 10-5 % biotin, 1% Glycerol) taken in 500 ml conical flask along with negative control (Pichia transformed with pPICK9K without insert) and were incubated at 28°C in a shaker incubator at 250 rpm until the culture reached an A600 of 4-5. The cells were harvested by centrifugation at 3,000 × g for 10 min at room temperature and the cell pellets were resuspended in required volume of either fresh YPM induction media (1%Yeast extract, 2%bacto peptone and 0.5 - 2.5% Methanol, buffered with 100 mM potassium phosphate buffer, pH 8.2) or Buffered Minimal Methanol medium-BMM (100 mM potassium phosphate, pH 8.2, 1.34% YNB, 4 × 10-5 % biotin, 0.5%- 2.5% methanol) so as to get an A600 of 3 in all. Several methanol concentrations ranging from 0.5 to 2.5% (0.5%, 1%, 1.5%, 2% and 2.5%) were tried in both media in order to choose the optimum concentration of methanol for induction in case of shaker flask culture. Incubation was continued at 29°C on an orbitary shaker (250 rpm) for four days. To sustain induction, required volume of methanol was added to every flask once in every 24 hour. Culture supernatants were collected at different time points ranging from 16-96 h (16 h, 23 h, 46 h, 72 h and 96 h) and were concentrated to 1/10 of its original volume using cellulose membrane with a pore diameter of 10 KDa (Millipore Corporation, USA) by centrifuging at 4000 g for 10-20 min at 4°C. Protease inhibitor cocktail (Amersco, USA) was added to the concentrated samples and the samples were stored at -80°C until all the time points were collected. Two clones with multiple copy integrants (Showing Geneticin resistance up to 750μg/ml and also resulted in intensive HA amplified PCR product) showing high expression of a recombinant protein were further selected for subsequent optimization experiments. The expression conditions viz methanol concentration (0.5-2.5%), type of medium (YPGy or BMGM), pH of the medium (6 - 8.5) and the time of harvest (16 h, 23 h, 46 h, 72 h and 96 h) were tested in order to get the intact HA protein expressed in abundance without any host specific proteolytic cleavage. The final protein obtained after optimization of the expression conditions obtained from cell culture supernatants were determined through Bradford assay against BSA standards . The protein samples were further analyzed by running them on 10% polyacrylamide gel electrophoresis (PAGE) both under denaturing and native conditions . The gels were subsequently stained with Coomassie Brilliant Blue R-250 (Sigma, USA). The expressed HA protein was confirmed through western blotting using rabbit anti H1N1 HA specific polyclonal antibodies (Genscript, USA) and goat anti rabbit alkaline phosphatase conjugated IgG (Sigma, USA) as primary and secondary antibodies respectively. The colour development was done either using BCIP/NBT solution (Sigma,USA) or H2O2/DAB substrate/chromogen (Sigma,USA).
Concentration of HA protein
The culture supernatants showing high level of secreted expression under optimal expression conditions were collected after methanol induction. The protein were concentrated ten times using 10 KD MWCO spin columns (Millipore,USA) by centrifuging at 4000 g for 20-30 min at 4°C.
FPLC purification of the Yeast derived HA protein based on size exclusion principle
The Yeast expressed protein recovered from the yeast culture supernatant was subjected to fast protein liquid chromatography (FPLC) using Akta explorer (Amersham, USA) employing previously equilibrated (in two column volumes of 20 mM Phosphate Buffered Saline,pH 7.2) Superdex 200 10/300 column (GE-Healthcare, USA). The Hemagglutinin protein of concentration 3 mg/ml diluted in 2.5 ml of Phosphate Buffered Saline (pH 7.0) was injected into the size exclusion chromatography column and the proteins with different sizes were eluted by monitoring the protein at 280 nm. The peaks obtained were compared with the known molecular weight marker proteins (GE-Healthcare, USA). Fractions corresponding to monomers and trimers of HA were collected separately for further use.
Analysis of the recombinant HA protein for trypsin cleavage
The yeast derived HA protein was checked for its cleavage into HA1 and HA2 fragments via trypsin digestion. To 10μg of yeast expressed HA protein, trypsin (100μg/ml stock made in PBS, pH7.2) was added to a final concentration of 1μg/ml and incubated at 37°C for one hour. The digested HA protein sample along with the negative control (HA protein undigested with trypsin) were run on 10% native PAGE and stained with coomassie brilliant blue.
Immunization of mice and rabbits with yeast derived H1N1HA recombinant protein and detection of serum antibodies through antibody capture ELISA
The animal studies experiments had an approval from the Institutional Animal Ethics Committee (IAEC) wide registration number 37/1999/CPCSEA and Institutional Biosafety committee (IBSC) wide reference no: IBSC/VIRO-01/05/TNA as per the institutional norms. The principles of good laboratory animal care were followed all through the experimental process. Eighteen healthy BALB/c mice 6-8 week-old were made into three groups with six animals in each group. The animals were found to be sero negative for the circulating H1N1 influenza. Two groups were immunized intramuscularly each with 50μg and 10μg of yeast derived H1N1 HA protein in Freund's complete adjuvant (FCA) (Sigma,USA) and the animals of the control group were immunized with the expressed product of negative control P. pastoris. Similarly two healthy adult male New Zealand White rabbits tested sero negative for H1N1 were immunized intramuscularly with 50μg each of yeast derived HA protein in combination with FCA for negative control, one rabbit was similarly immunized with FCA alone. After two weeks, animals were boosted once with the same amount of protein in combination with Freund's Incomplete adjuvant (FIA) (Sigma, USA). Following two weeks of the booster dose, blood samples were collected either from retro-orbital route (In case of mice) or marginal veins (rabbits) and the sera were separated. Antibody capture ELISA was performed to determine the antibody titre against the H1N1HA protein . Briefly, the yeast expressed H1N1 HA protein was coated overnight onto Nunc polystyrene microtitre plates (300 ng/well in 0.1 M sodium bicarbonate, pH 8.0) and the non reacted sites were blocked using 3% BSA. The captured proteins were reacted first with the immunised mice and rabbit sera and were subsequently incubated with their respective anti-species secondary HRP conjugates (Sigma, USA) for 1 hour. Enzymatic colour development was done using TMB/H2O2 chromogen/substrate solution. The samples showing the OD values twice that of negative serum were considered to positive and were used in determining the titre of the antibody.
Culturing H1N1 virus and determination of its Hemagglutination (HA) titre and virus quantification through Plaque assay
The H1N1 virus isolate from clinical sample in Bangalore, India during 2009 outbreak was a kind gift from Dr.V.Ravi, NIMHANS, Bangalore, India. The H1N1 virus was propagated using Madin Darby Canine Kidney (MDCK) cell lines as described earlier [45, 49]. Hemagglutination assay was performed on the H1N1 virus stock using chicken RBC. Briefly, chicken RBCs were separated from the whole blood and washed three times in PBS (pH7.2). Fifty micro litres of 0.5% RBC suspension (v/v in 1% PBS) was added to 50μl serially diluted H1N1 influenza virus in U-bottom 96 well plates. The plates were incubated at room temperature for one hour and were observed for the formation of button or mat within the wells.
For quantifying the H1N1 virus, plaque assay was performed by serially diluting the virus stock on MDCK cells in six well plates. The plaques formed after three days of post infection were counted and the titre of the virus stock was determined. The H1N1 virus stock with known HA titre and virus concentration was used for subsequent experiments. The culturing of the virus, determination of Hemagglutination (HA) test and plaque assay were performed in Bio safety level 3 (BSL-3) laboratory.
Hemagglutination Inhibition (HI) activity of H1N1HA immunised mice and rabbit sera
Two fold dilutions of immunised/control mice and rabbit sera were made in U-bottom 96-well micro titre plate. Four Hemagglutination units (HAU) of influenza virus were added in each well and the virus-serum mixture was incubated for 30 minutes and 0.5% suspension of chicken RBC (in PBS pH7.2) were added and mixed by agitation. The chicken RBCs were allowed to settle for one hour at room temperature and HI titres were determined by the reciprocal value of the last dilution of the sera which completely inhibited the Hemagglutination of chicken RBCs.
In-vitro neutralisation activity of H1N1HA immunised mice and rabbit sera
Serum samples were heat-inactivated at 56°C for 30 minutes. Two-fold serial dilutions from 1:16 to 1:1024 were prepared in virus diluent (MEM with L-glutamine) containing no serum or antibiotic/antimycotic solution. Serially diluted serum was challenged with an equal volume of the H1N1 virus, previously titrated to give 100 pfu in 250 μl of virus dilution. The virus control of the experiment contained the virus diluted in the virus diluents without serum. The serum/virus mixtures were incubated at 37°C, 5% CO2 for one hour. MDCK cell monolayers, prepared in six well plates were infected with 500 μl/well of the serum/virus mixture. Plates were incubated at room temperature for one hour. The supernatants were completely aspirated out from the wells and the wells were overlaid with 1.5% low melting point agarose (Sigma,USA) prepared in 2X MEM containing 5 μg/ml trypsin. Plates were incubated for plaque formation at 37°C, 5% CO2 for 3 days and the wells were stained with 0.2% crystal violet solution (made in 30% ethyl alcohol). The plaques formed were counted and neutralisation activity of the immune sera was assessed by comparing the plaque numbers obtained from that of negative control serum. The highest dilution of the sera that showed more than 50% reduction in plaque number than that of negative control is considered as the neutralising titre.