1. ESID Prague Spring Meeting 2009
FUNCTIONAL INVESTIGATION OF ANTIBODY PRODUCTION IN COMMON VARIABLE IMMUNODEFICIENCY (CVID) PATIENTS UNDER IMMUNOGLOBULIN SUBSTITUTION
Zita Trávníčková
Department of Clinical Immunology and Allergology of St. Anne´s University Hospital and Medical Faculty of Masaryk University Brno
ESID Prague Spring Meeting 2009

2. ELISPOT assay in PID Detection of specific antibody
functional investigation
of B cells
Detection of specific antibody
production on B cell level
independent on substitution Ig therapy

3. EXPERIMENTAL DESIGN SUBJECTS VACCINATION
37 CVID patients (15 males, 24 females, age range years)
81 healthy donors (28 males, 53 females, age range years)
VACCINATION
ALTEANA (Sevapharma a.s., Prague, Czech Republic)
PNEUMO 23 (Sanofi Pasteur, Lyon, France)
ELISPOT (isolated MNC of peripheral blood, calculated on CD19+)
day 0, day 7, weeks 4-11

4. HEALTHY DONORS
Vaccination with PROTEIN antigen
50 controls (16 males, 34 females, age range 22 – 72 years)
Vaccination with POLYSACHARIDE antigen
10 controls ( 4 males, 6 females, age range 15 – 46 years)
Vaccination with PROTEIN and POLYSACHARIDE antigen
21 controls (8 males, 13 females, age range 14 – 50 years)

5. FROM 4 TO 11 WEEKS AFTER VACCINATION
HEALTHY DONORS
BEFORE VACCINATION
DAY 7 AFTER VACCINATION
FROM 4 TO 11 WEEKS AFTER VACCINATION
MED
MIN
MAX
TAT
SFC/106 BL
IgG
10 367
330
86 747
IgA
482 24
8 876
IgM
PCP
4 133
812
9 540
35 965
5 739
3 869
95 571
52 994
TAT + PCP
9 008
964
45 268
556 31
9 707
3 736
1 004
76 880
35 200
3 200
10 201
2 165
44 480

6. CVID PATIENTS
Vaccination with PROTEIN and POLYSACHARIDE antigen
37 patients (15 males, 24 females, age range years)

7. 2% switched memory B cells 2% switched memory B cells
EUROclass B-cells
(Blood 2008)
> 1% B cells
 group B+
1% B cells
 group B-
2% switched memory B cells
 group smB+
2% switched memory B cells
 group smB-
 10% CD 21low B cells
group smB+21lo
 10% CD 21low B cells
group smB-21lo
< 10% CD 21low B cells
group smB+21norm
< 10% CD 21low B cells
group smB-21norm

8. EUROclass B cells CVID group B+ 37 patients group B- group smB+
group smB+21lo
10 patients
group smB-21lo
12 patients
group smB+21norm
6 patients
group smB-21norm
9 patients

9. ELISPOT smB+21norm SmB+21lo CVID BEFORE VACCINATION PROTEIN antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7 8 9 10
smB+21norm
PROTEIN
Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6

10. ELISPOT smB+21norm SmB+21lo CVID DAY 7 AFTER VACCINATION 713 2 562 659
PROTEIN
antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7
713 8 9
2 562
659 10
104
smB+21norm
PROTEIN
Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3
185 4
231 5
407 6

11. ELISPOT smB+21norm SmB+21lo CVID 4-11 WEEKS AFTER VACCINATION PROTEIN
antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7 8 9 10
smB+21norm
PROTEIN
Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6

12. ELISPOT SmB-21lo smB-21norm CVID BEFORE VACCINATION PROTEIN Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7 8 9 10 11 12
smB-21norm
PROTEIN
Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7 8 9

13. ELISPOT smB-21norm SmB-21lo CVID DAY 7 AFTER VACCINATION 344 PROTEIN
Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7 8
344 9 10 11 12
smB-21norm
PROTEIN
Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7 8 9

14. ELISPOT SmB-21lo smB-21norm CVID 4-11 WEEKS AFTER VACCINATION PROTEIN
Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7 8 9 10 11 12
smB-21norm
PROTEIN
Antigen
SFC/106 B cells
POLYSACCHARIDE
IgG
IgA
IgM 1 2 3 4 5 6 7 8 9

15. CONCLUSIONS
The ELISPOT assay is sensitive functional test for determination of specific antibody production in PID patients under substitution immunoglobulin therapy.
In well-defined CVID patients almost no detectable peripheral B-cells producing specific IgG, IgA and IgM antibodies agains Tet. Tox. and PCP were observed.
If the IgG response was detectable after Tet. Tox. vaccination most patients but not all were characterized as smB+21norm according to EUROclass.

16. 2009
SPECIAL THANKS TO CO-WORKERS Department of Clinical Immunology and Allergology St. Anne´s University hospital and Medical Faculty of Masaryk University in Brno
prof. MUDr. Jiří Litzman, CSc. MUDr. Vojtěch Thon, Ph.D. Mgr. Marcela Vlková, Ph.D.
1910
St. Anne´s University Hospital Brno