Department of Physics, Chemistry and Biology (IFM)

Animals and Immunization:

In both the experiments, serum samples and lung washes of the mice which were immunized intranasally with vaccine were obtained from Royal technical High School, Stockholm. In the first experiment six groups (n=8 mice per group) of BALB/c mice were immunized with Influenza A split virus vaccine with endocine^TM adjuvants supplemented with stabilizators. All the mice used in this study were administered with endocine^TM adjuvants at concentration of 20mg/ml. Stabilators along with the vaccine and adjuvants have been used to immunize mice of group 4, 5 and 6 while the mice from groups 1 , 2 and 3 were immunized only with vaccine and adjuvants but with different formulation of adjuvants. A naïve group (group 7) of mice has been included in this experiment which was not immunized. The mice were sedated before each immunization. They were immunized intranasally with 10µl of vaccine (5µl in each nostril) containing a dose 1.5µg of HA (per mouse/adjuvant) once for every 3 weeks (day 0, 28, 49) for 3 times. Blood samples were collected on day 42 and day 56. After the final bleeding, the mice were sacrificed and the lungs were collected and frozen for serological analysis. The concentration of the adjuvants along with the stabilizators administered with the vaccine is shown in table 1. All the experiments on mice in this study are approved by ethical committee at karolinska institutet.

In the second experiment 4 groups (n=6 mice per group) of BALB/c mice were immunized with vaccine containing a dose 4.5µg of HA via different routes of administration. The concentration of the adjuvants used with the vaccine and type of administration in each group is shown in table2.

Detection of Specific antibodies using Enzyme-linked Immunosorbent Assay:

96-well Elisa plates (Nunc Maxisorp, Odense, Denmark) were coated with Recombinant Hemagglutinin (HA) using a trivalent vaccine (A/California/7/2009(H1N1), A/Perth/16/2009(H3N2), B/Brisbane/60/2008),VACCINE MOT INFLUENSA, Lotnr: G9758-1, Sanofi Pasteur MSD, Bryssel, Belgien) at a concentration of 0.5µg mL^-1 in carbonate coating buffer (0.05M Na₂CO₃, pH 9.5-9.7) and a volume of 100 µL of antigen/coating buffer mixture was added to each well and incubated overnight at room temperature.

Plates were washed three times with saline buffer (0.9% Nacl containing 0.05% of Tween-20). For IgG analysis, the plate was first blocked by adding 100 µL of blocking buffer containing PBS and 5% dry milk (Lotnr: 20243, Scharlau) to each well and incubated for 1h at +37 ^oC. Later the plate was washed for three times with saline buffer and the diluted serum samples were added at a volume of 100 µL to each well followed by incubation at +37 ^oC for 1.5 h. This was followed by another washing for five times with saline buffer. Biotinylated Goat Anti-Mouse IgG (H+L)-HRP conjugate (Biorad) diluted at 1:3000 was added at 100µL to each well and incubated again for 1.5 h at +37 ^oC after which the plate was washed four times with saline buffer. Goat-anti-subclass IgG’s (Sigma Aldrich) diluted to 1:1000 were used in the analysis of IgG1, IgG2a, and IgA. The conjugate, Anti-goat IgG Peroxidase (Sigma Aldrich) was used at a dilution of 1:20000 for sub class analysis. 2 mg of O-phenylenediamine tablet (Sigma) was dissolved in 20 mL of substrate buffer (0.1 M Sodium Citrate buffer, pH 5.0 activated by 0.03% H₂O₂) was added at 100 µL per each well and the plate was allowed to develop at room temperature in dark for 30 min. The substrate reaction was then stopped using 2M H₂SO₄ added at a volume of 100µL to each well and the plate was read at 490 nm in a micro plate reader.

Avidity Elisa:

The plate was coated with recombinant hemagglutinin and blocked with 5% milk in the same way as mentioned above. 100 µL of the serial dilutions are added to the plate as duplicates and incubated at +37 ^oC for 1.5 h. The plate was then washed once with saline buffer. Then, one half of the plate (one set of duplicates) was washed once with saline buffer (5 minutes) while the other half of the plate was washed with Urea (8M, pH 8.0) (5 minutes). The whole plate was washed thrice with the saline buffer and tapped dry. Conjugate was added and the plate was developed in the same procedure as described above. A difference in the antibody avidity between the wells with 8.0M urea and wells with saline buffer was calculated to give an avidity index (AI) using the formula:

Avidity Index (AI) = (reaction with 8M urea/reaction with saline buffer)

Lung washes

The lungs of the mice that were immunized by the vaccine were obtained for examination of total IgA and IgG against Influenza A antigens. The lungs were thawn and dissolved with 0.4 mL of sterile H₂O containing a protease inhibitor (5x) (Sigma) and stored frozen until used.  Plates were coated with Recombinant Hemagglutinin and blocked as described above. 100 µL of the undiluted and ¼ diluted (PBS- 2.5% dry milk, 0.05% Tween 20) lung wash samples were added to each well according to the ELISA test protocol and the plate was sealed. It was then left for incubation overnight (16-20 h) at room temperature. The antibodies and the conjugate were later added according to the method described above.

RDE Treatment of the Sera

The samples were pre-treated with receptor destroying enzyme (RDE), chicken and guinea pig red blood cells to remove hemagglutination inhibitors and unspecific agglutinins. Chicken blood was also used in order to check the cross reactivity of the vaccine with Brisbane strain. The sera to be used were mixed with RDE and incubated overnight in a water bath at a temperature of 37 ^oC which is followed by incubation on the next day by changing the temperature to 56 ^oC for 30 min. Then required amount of 0.9% NaCl was added to the samples. Required amount of PBS was added to blood, mixed well (10%) and centrifuged. This was repeated until a clear suspension (supernatant) was obtained. Later, the washed red blood cells were added to the tubes with sera (end dilution 1:10) and incubated for 1 h (vortexing for every 15 min). Later the tubes were centrifuged at 5 min at 2000 rpm. The sera (supernatant) were collected into a new tube and were used for Hemagglutination Inhibition assay (HIA) and Neutralization assay.

Hemagglutination Assay for determination of Virus titer

1% of Guinea pig blood was prepared by mixing with PBS and washed until a clear suspension was obtained. V-shaped 96 well plate were chosen and labeled. 50 µL of PBS was added to all the wells that were used. 50 µL of virus was added to the first row of wells. The virus was then serial diluted to the next following wells. A control was included at last row each dilutions which only contained PBS and red blood cells. This is followed by addition of 50 µL of blood to all the wells. The plate is then sealed, gently tapped for mixing and left for incubation for 1 hour at room temperature. The dilution at which no spot of RBC was observed is determined the titer of virus for hemagglutination.    

Hemagglutination Inhibition Assay

V-bottomed 96 well plates were chosen for carrying the HIA. These plates were labeled according to the protocol. The samples chosen for the assay were prepared in duplicates. 25 µL of PBS was added to all the wells except for first row.  50 µL of the RDE treated sera (dil 1:10) was added to the first row of wells as duplicates. 30µl of the sera was titrated to the second row of wells containing PBS and so on until the row G. The last row on the plate was used as a control containing only PBS and RBC. A virus dilution that is equivalent to 4 HA units was prepared and 25 µL of it was added to each well except the last row i.e. control. The plate was tapped gently for mixing the virus and sera and left for incubation for 15 min. Later, 50 µL of 1% Guinea pig blood was added to all the wells on the plate and left for incubation for 2 h. The dilutions at which the wells show an intense red spot (RBC) were noted as the titer to inhibit virus agglutination.

Determination of tissue culture infectious dose (TCID50) and Neutralization Assay

Preparation of MDCK cells

MDCK cells (Madine darby kidney cells) were grown in cell culture flasks in culture medium (RPMI 1640  plus 8% FCS, 4mM Na-pyruvate, 1% PEST, 0.5 mL MF and L-glutamine) in incubator at 37 ^oC and 5% CO₂ until a good concentration (75-95% confluence) is obtained. On the day of experiment the culture medium was discarded and the monolayer of cells was washed thrice with PBS. 1.5 mL of mixture of solution containing trypsin and serum free RPMI medium (4 mM Na-pyruvate, 1% PEST, 0.5 mL MF and L-glutamine) in a dilution of 1:2 was added to the flask and placed in incubator at 37 ^oC and 5% CO₂ until the monolayer got dislodged from the surface of the flask (approximately 5-10 minutes). The flask was gently tapped at the sides to remove all the cells. The flask was then rinsed once with 8 mL of cell culture medium which was then transferred into a new 50 mL tube. The tube was centrifuged at 1500G for 7 minutes resulting in the formation of cell pellet at the bottom of the tube. The supernatant was discarded and the cells were re suspended by tapping at the bottom. 1 mL of cell culture medium was added to the tube. A separate 1:10 dilution of a mixture containing cell suspension and Trypan blue stain was prepared and placed on the hematocytometer for observation under the microscope to determine the number of cells per ml. A 96-well microtiter plate was taken and each well was seeded with MDCK cells at a concentration of 10⁴cells/200 µL. The plate was left in the incubator at 37 ^oC and 5% CO₂ for 3 days.

 Addition of virus (TCID50)

On the day of infection, virus dilutions containing trypsin at concentration of 5µg mL^-1 were prepared using the serum free RPMI medium with a dilution factor of 10 (10^-2 to 10^-7). The medium in the microtiter plate with MDCK cells was removed and each well of the plate was washed thrice with 200 µL of PBS (the cells were not allowed to dry). 50 µL of each virus dilution was added to each well according to the protocol (the row of wells in the borders of the plate were not used and the virus dilutions were added as sextuplets). The virus was allowed to infect the cells in the plate by incubating at 37 ^oC and 5% CO₂ for 2 h. After adsorption, the virus medium was removed from the plate with the cells and 200 µL of serum free RPMI medium containing 5µg/ml of trypsin was added to each well and left in incubator at 37 ^oC and 5% CO₂ for 3-4 days.  

 Addition of Virus and sera mix (Neutralization assay)

On the day of infection dilutions of the inactivated serum samples and the virus were prepared using the serum free medium. The starting dilution of the serum used was 1:50. U-bottomed Ninety six well plates were taken and two fold serial dilutions of the serum samples were added to all wells as duplicates according to the protocol. Equal volume of virus was added to all the wells. The plate was sealed, gently tapped (for proper mixing) and incubated for 1 h at a temperature of 37 ^oC and 5% CO₂. The plate with the grown MDCK cells was taken and the medium was discarded and washed thrice with PBS as described above. 50 µL of the sera and the virus mix (from U bottomed plate) was added to wells of the plate with MDCK cells according to the protocol and incubated for 2 h at 37 ^oC and 5% CO₂. After 2 h the plate was removed from incubator and the virus and sera mix was discarded. 200 µL of serum free RPMI medium was added to each well and incubated in the incubator at 37^oC and 5% CO₂ for 3-4 days.

 Fixation of cells

The plate was taken after 3-4 days and centrifuged for 10 minutes at 1500*g. The cell medium was removed. 100 µL of ice cold acetone was added to each well and left for 10 minutes at room temperature. Acetone was later removed and the plate was left to air dry in the flow hood at room temperature.

 Elisa

The plate was then washed for four times with saline buffer. 100 µL of 5% dry milk in PBS was added to each well and left in incubator for one hour at 37 ^oC. The plates were flicked empty and 100 µL of mouse serum, anti-Influenza (1:5000) diluted in 2.5% dry milk/PBS/0.05% Tween 20 was added to each well. The plate was allowed to incubate for 90 minutes at 37 ^oC. The plate was then washed four times with saline buffer followed by an addition of Anti human IgG (1:10000) (DAKO, Glostrup, Denmark) in dilution buffer at 100 µL/well. The plate was later developed and read as described above. TcID50 was calculated using REED-MUENCH method.

Statistical Analysis

The total data of IgG, IgG1, IgA and IgG2a antibody titers in BALB/c mice were analyzed using GraphPad version 5.01 for Windows, Graphpad software, San Diego, CA, USA, www.graphpad.com. The median serum titers of different groups were compared using ANOVA. A probability value (p) <0.05 was declared statistically significant.