Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT01002716 |
| Other study ID # |
TASMC-09-OE-0453-CTIL |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
Phase 4
|
| First received |
October 15, 2009 |
| Last updated |
October 26, 2009 |
| Start date |
October 2009 |
| Est. completion date |
April 2010 |
Study information
| Verified date |
October 2009 |
| Source |
Tel-Aviv Sourasky Medical Center |
| Contact |
Ori Elkayam, MD |
| Phone |
97236973668 |
| Email |
orie[@]tasmc.health.gov.il |
| Is FDA regulated |
No |
| Health authority |
Israel: The Israel National Institute for Health Policy Research and Health Services Research |
| Study type |
Interventional
|
Clinical Trial Summary
The efficacy of vaccination against influenza in patients with rheumatoid arthritis has been
assessed using humoral response. However, the cellular immunity is another important pathway
of response to vaccination. The purpose of this study is to evaluate the degree of cellular
immunity response to influenza vaccination. Patient with rheumatoid arthritis and healthy
controls will participate in this study , will undergo a clinical evaluation the day of
vaccination and 4 weeks after. The humoral and cell immunity response will be assessed the
day of vaccination and 4 weeks later
Description:
Sixty patients with RA and 30 healthy , aged matched controls will participate in the study.
After signing informed consent, all subjects will be vaccinated with the inactivated split
virion vaccine which will be recommended by the WHO next fall.
Patients will be evaluated at weel 0 and 6 weeks later. Clinical evaluation will be based on
the Disease Activity Score 28 (DAS 28) which includes number of swollen and tender joints,
visual global evaluation of the physician , ESR and CRP Blood with be collected on the day
of vaccination and 6 weeks later.
Preparation of peripheral blood mononuclear cells Blood samples will be obtained prior to
and 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) will be isolated by
centrifugation of heparinized blood on a Lymphoprep gradient (Nycomed, Oslo, Norway). Cells
will be washed twice in tissue culture medium RPMI (RPMI1640-Hepes with Glutamax-I, GIBCO
BRL, NY, USA) and resuspended to a concentration of 2 106/ml in RPMI 1640 supplemented with
10% heat inactivated fetal bovine serum (FBS) (GIBCO BRL, NY, USA).
ELISPOT assay The 96-well PVDF (polyvinylidene difluoride, Millipore Corp., MA, USA) plates
will be prewet with 70% EtOH, then coated with 100 l/well of anti-human IFN- monoclonal
antibody (mAb) 1-D1K , diluted to 15 g/ml in sterile filtered phosphate-buffered saline
(PBS) . The plates will be kept overnight at 4°C. A total of 100 000 PBMC plus 5 l/well (1
g/ml) peptides will be added to each well in duplicate for each condition and incubated for
24 h at 37°C in 5% CO2. After incubation the plates will be washed with PBS then 100 l of
biotinylated mAb (7-B6-1-biotin, Mabtech AB), diluted in filtered PBS with 0.5% FBS to the
concentration 1 g/ml were added to each well. The plates will be incubated 2 h at room
temperature, washed with PBS and then incubated with 100 l/well of streptavidine-alkaline
phosphatase (Mabtech AB) diluted 1-1000 in PBS with 0.5% FBS for 1 h at room temperature.
After washing, cells will be developed by adding 100 l/well of colorimetric substrate
(BCIP/NBT-plus, Mabtech AB) and then counted in an ELISPOT reader. Each spot was produced by
a single IFN- secreting T-cell-spot-forming cell (SFC). All tests were performed in
duplicate and the mean values were calculated. A response will be considered to be positive
when the number of spots in the wells with peptide-stimulated cells, after subtraction of
the background (wells without peptide stimulation), will be at least two-fold greater than
the number of background spots14 and more than 20/106 SFC.
Detection of intracellular cytokine production by flow cytometry The intracellular staining
method will be used for identifying the phenotype of INF- producing T cells activated by
peptide stimulation. The staining will be performed using the protocol described by Rauser
et al.15 PBMC were stimulated for 6 h with 1 g/ml influenza peptides. Brefeldin A (10 g/ml,
Sigma-Aldrich, St Louis, MO) will be added for the last 4 h of the incubation. A positive
control will be obtained by stimulating the cells with 0.5 g/ml PMA (Phorbol 12-myrestate
13-acetate) and 1 g/ml Ionomycin (both from Sigma-Aldrich, St Louis, MO, USA). The cells
will be permeabilized and stained with fluorochrome-labeled anti-CD3, anti-CD4 or anti-CD8
and anti-IFN- antibodies (Becton Dickinson, San Jose, CA, USA). Samples will be analyzed by
flow cytometry using FACSCalibur (Becton Dickinson). The positive limit for this test was
0.05% of IFN producing T cells.
Proliferation of specific CD4+ and CD8+ Cellular response will be evaluated by
simultaneously analyzing proliferation of specific T CD4+ and CD8+ lymphocytes upon
stimulation with antigens of the three virus strains present in the vaccine and production
of activation Th1 cytokines by the same cells.
Briefly, to simultaneously analyze proliferation and cytokine production by influenza
specific T cells, peripheral blood mononuclear cells will be obtained at T0 and T1 from each
donor, stained with the fluorescent nuclear dye carboxyfluorescein succinimidyl ester (CFSE;
Molecular Probes) and cultured in duplicate in the presence of peptides of HA from each of
the virus strains contained in the influenza virus vaccine used in the study or control
peptide for 72 h. At the end of incubation cultures will be stopped with brefeldin, cells
resuspended, stained with fluorochrome-conjugated anti-CD8 and anti-interferon-γ (IFN-γ)
monoclonal antibodies (Becton&Dickinson) and analyzed by mean of a six colour flow-cytometer
(FACSCanto-Becton&Dickinson) and FACS DIVA software . As at every cell division the nuclear
fluorescence is halved, proliferating cells fall in the left quadrants of the cytogram, the
more divisions the less fluorescence. IFN-γ producing cells fall in the upper quadrants of
the cytograms. Cells that upon specific influenza peptide or control peptide stimulation
both proliferate and actively produce IFN-γ (Th1 cytokine) fall in the upper left quadrant
of the cytogram. They were measured as frequency (proliferating and IFN-γ-producing
cells/total T CD4+ or CD8+ lymphocytes).
Haemagglutination inhibition test The immunogenicity of the vaccine was tested by
Haemagglutination inhibition (HI) test.
Influenza virus has two important surface glycoproteins: the haemagglutinin (HA) and the
neuraminidase (NA). Antigenic classification and subtyping of influenza viruses is based on
these two glycoproteins. HA plays a key role in virus cell entry by binding to cell surface
receptors, which are found also on red blood cells of certain species. Binding to red cells
results in haemagglutination, which can be observed as a carpet of agglutinated red cells at
the bottom of a tube or microtitre well. In the HI test, antibody directed against the viral
haemagglutinins block the virus from binding to the blood cells and thus inhibits the
haemagglutination reaction.
The pre- and post immunization HI antibodies were tested at the Central Virology Laboratory
of the Israeli Ministry of Health using the HI test according to a standard WHO procedure
16. Sera were separated, code labeled, and stored at -20°C until tested. Sera were treated
with receptor destroying enzyme cholera filtrate to remove non-specific inhibitors, and with
Turkey red blood cells to remove non-specific agglutinins. The treated sera were tested by
HI test against the three antigens included in the vaccine: A/California (CAL), B/Shangai
(SHAN) and A/New Caledonia (NC). The working dilution (test dose) of each antigen contained
four haemagglutinin units in 25 µl of antigen. Test doses were diluted in phosphate buffered
saline (PBS) and added to serial dilution of antiserum. The haemagglutinin inhibition titer
was determined as the highest dilution of serum that completely inhibits haemagglutination
of red blood cells.
The titer of an antiserum not showing any inhibition was recorded as <10. Humoral response
was defined as either a fourfold or more rise in titer, or a rise from a non-protective
baseline level of <1/40 to 1/40 in HI antibodies four weeks after vaccination 17,18.
Geometric mean titers of antibody were calculated to assess the immunity of the whole group.
Primary Endpoint of the study : the proportion of cells producing Interferon gamma in
Patients with rheumatoid arthritis and controls Secondary Endpoint: Safety of the vaccine
