Effect of Tocilizumab to the Cellular Immune Response to Influenza Vaccine
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|ClinicalTrials.gov Identifier: NCT01980836|
Recruitment Status : Unknown
Verified November 2013 by Tel-Aviv Sourasky Medical Center.
Recruitment status was: Not yet recruiting
First Posted : November 11, 2013
Last Update Posted : November 11, 2013
|Condition or disease||Intervention/treatment||Phase|
|Rheumatoid Arthritis Cellular Immune Response||Biological: Seasonal influenza vaccine||Phase 4|
Inclusion criteria :
- RA patients
- Above the age of 18
- Treated with tocilizumab at least 3 months
Design of the study :
- Patients and controls will be vaccinated against sesaonal influenza. Blood will be taken the day of vaccination and 4 weeks after.
PBMC isolation The PBMCs will bere isolated from heparinized venous blood by density gradient centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway) immediately after blood was drawn. The cells will be counted and suspended in RPMI 1640 supplemented with heat-inactivated 10% fetal calf serum, penicillin 100U/ml, streptomycin 0.1 mg/ml, and 2 mM L-glutamine (Biological Industries, Israel).
IFN-gamma secretion from PBMCs IFN-gamma secretion levels will be measured by ELISA in the supernatants of PBMCs that will be stimulated with either an influenza antigen mixture or with SEB, or left untreated.
Granzyme B activity assay The PBMCs will be cultured at 0.5 ml/well in 48-well plates (1.8X106 cells/well) and stimulated with an influenza antigen mix and SEB as described above. The cells will be lysed in lysis buffer (150mM NaCl, 15mM Tris, 1% Triton x100), then stored at
-760C. Granzyme B activity will be measured according to the protocol described by Gijzen et al. . Briefly, frozen cell lysates will be subjected to three freeze/thaw cycles to enable the release of Granzyme B. Recombinant Granzyme B standards (Enzo Life Sciences International, Inc., PA) and cell lysates will be added in duplicate to a 96-well plate (20 µl/well). The reaction will start upon the addition of 80 μl of substrate solution containing 400 μM of Ac-IEPD-pNA substrate (Calbiochem, Darmstadt, Germany) in assay buffer (100mM HEPES pH 7.5, 10% (w/v), sucrose, 0.1% (w/v) CHAPS, and 10 mM DTT (Sigma Aldrich, Rehovot, Israel). The plate will be sealed, covered and incubated in a dark humidified chamber at 370C for 20 h. After incubation, the plate will be read at 405 nm. Granzyme B units will be calculated using a 4th order polynominal curve with a log (concentration)−log (absorbance) plot, and corrected for protein concentrations by the BCA protein assay (Thermo Scientific, IL).
The humoral response The antibody response will be measured by the HI test according to a standard WHO procedure as previously described . The titer of an antiserum not showing any inhibition will be recorded as 1/10. Humoral response is defined as either a fourfold or greater rise in the titer of HI antibodies, or a rise from a non-protective baseline level (<1/40) to 1/40). Geometric mean titers of antibodies wull be calculated to assess the immunity of the whole group.
|Study Type :||Interventional (Clinical Trial)|
|Estimated Enrollment :||40 participants|
|Intervention Model:||Parallel Assignment|
|Masking:||None (Open Label)|
|Official Title:||The Effect of Tocilizumab on the Cellular Immune Response to Seasonal Influnza Vaccine in Patients With Rheumatoid Arthritis|
|Study Start Date :||November 2013|
|Estimated Primary Completion Date :||February 2014|
|Estimated Study Completion Date :||February 2014|
Active Comparator: Seasonal influenza vaccine in tocilizumab treated RA patients
RA patients treated with tocilizumab
Biological: Seasonal influenza vaccine
Active Comparator: Seasonal influenza vaccine to Healthy controls
Healthy controls will be vaccinated against seasonal influenza
Biological: Seasonal influenza vaccine
- Cellular response [ Time Frame: 4 weeks ]As described in the study design using the Interferin gamma and granzyme B levels
- Humoral response [ Time Frame: 4 weeks ]Using an hemaglutination inhibition test as described in the study design
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT01980836
|Contact: Ori Elkayam Elkayam, M.D||97236973668||ORIE@TLVMC.GOV.IL|
|Tel Aviv Medical Center|
|Tel Aviv, Israel, 64239|
|Contact: Ori Elkayam, M.D 97236973668 ORIE@TLVMC.GOV.IL|
|Sub-Investigator: Uri Arad, M.D|