Effect of Tocilizumab to the Cellular Immune Response to Influenza Vaccine

Tel Aviv Sourasky Medical Center

Status and phase: Unknown; Phase 4

Conditions: Cellular Immune Response; Rheumatoid Arthritis

Treatments: Biological: Seasonal influenza vaccine

Study type: Interventional

Funder types: Other

Identifiers

NCT01980836

TASMC-13-OE-245-CTIL

Details and patient eligibility

About

Patients with rheumatoid arthritis (RA) are prone to respiratory infections and therefore recommmended to receive vaccination against seasonal influenza. We and others have shown a relatively preserved humoral response to vaccination in RA patients. However, the cellular response as well as the effect of biologics such as tocilizumab on the cellular response has not been weel studied. The purpose of this study is the evaluate the effect of tocilizumab on the cellular immune response to influenza vaccine in patients with RA in comparison with healthy controls

Full description

Inclusion criteria :

• RA patients
• Above the age of 18
• Treated with tocilizumab at least 3 months

Exclusion criteria:

-egg allergy

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. [13]. 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 [14]. 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.

Enrollment: 40 estimated patients

Sex: All

Ages: 18+ years old

Volunteers: Accepts Healthy Volunteers

Inclusion criteria

• Rheumatoid Arthritis patients treated with Tocilizumab for at least 3 months
• Above the age of 18

Exclusion criteria

• Allergy to eggs

Trial design

Primary purpose: Prevention

Allocation: Non-Randomized

Interventional model: Parallel Assignment

Masking: None (Open label)

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40 participants in 2 patient groups

Seasonal influenza vaccine in tocilizumab treated RA patients

Active Comparator group

Description:

RA patients treated with tocilizumab

Treatment:

Biological: Seasonal influenza vaccine

Seasonal influenza vaccine to Healthy controls

Active Comparator group

Description:

Healthy controls will be vaccinated against seasonal influenza

Treatment:

Biological: Seasonal influenza vaccine