1. Rome, Italy - March 18-19, 2005 masque.... Humoral immune status: Comparison of various serological assays Catherine Sadzot-Delvaux Laboratory of Fundamental Virology Pathology, B23 4000 Liège BELGIUM

2. Rome, Italy - March 18-19, 2005 masque.... Humoral immune status: Comparison of various serological assays Immunity to VZV: –Complex –Not fully understood –Natural immune response (NK cells,…) –Specific immune response Humoral immunity: antibodies against glycoproteins, nucleocapsid and tegument proteins. Cellular immunity: mainly Th1.

3. Rome, Italy - March 18-19, 2005 masque.... Serodiagnosis is very useful –for verifying the immune status prior to vaccination of healthy, at high-risk adults without history of chickenpox –for assessing the immunogenicity of varicella vaccine and assuring the follow-up of vaccination studies. Humoral immune status: Comparison of various serological assays

4. Rome, Italy - March 18-19, 2005 masque.... Humoral immune status: Comparison of various serological assays Specific antibodies can be detected by: –neutralization –complement fixation –enzyme-linked immunosorbent assays ELISA gpELISA –immunofluorescence FAMA (Fluorescent antibody to membrane antigen) IFAT (Indirect fluorescence antibody test) –latex agglutination (LA)

5. Rome, Italy - March 18-19, 2005 masque.... Serological assays: ELISA (Enzyme-linked immunosorbent assay) Solid-phase enzyme immunoassay Antigen = extract of VZV-infected cells (ELISA) Output: optical density Simple and automated Adaptable to detect IgA, IgM or IgG Suitable for small- and large-scale testing  The most frequently used assay in particular in the follow-up of vaccination studies

6. Rome, Italy - March 18-19, 2005 masque.... Serological assays: ELISA (Enzyme-linked immunosorbent assay)BUT Lacks sensitivity and /or specificity Can be optimized if the preparation of antigens is optimized: the purer the antigens, the more sensitive and the more specific is the assay. Determination of class-specific antibodies is sometimes difficult –Large amounts of IgG that interfere with less frequent IgM –False-positive frequently observed for IgM due to rheumatoid factor (RF) that can be produced in viral infections and interfere with IgM assays.

7. Rome, Italy - March 18-19, 2005 masque.... Serological assays: VIDAS VZV (BioMerieux/Vitek) Rapid: VIDAS VZG: 40minBUT Requires an automate Some equivocal results (23/625) even after retesting but no possibility to control since all steps performed in an automate

8. Rome, Italy - March 18-19, 2005 masque.... Serological assays: gpELISA Modified version of the « classical » ELISA Antigen: purified VZV glycoproteins 4x more sensitive than ELISA Frequently used in post-vaccination studies.  Clinically relevant marker of functional immunity BUT Not commercialized

9. Rome, Italy - March 18-19, 2005 masque.... Serological assays: gpELISA 6 weeks after immunization –99% positivity (Mean titer:12.9 gpELISA Units) –Antibody titers correlate with levels of neutralizing antibody and the induction of cell-mediated immunity –Estimated vaccine efficacy: 95.5% if 6-week post- vaccination titer > 5gpELISA 83.5% if 6-week post- vaccination titer < 5gpELISA –3.5 x more likely to develop breakthrough varicella –More lesions Li S, et al. Pediatr.Infect.Dis J 2002;21:337

10. Rome, Italy - March 18-19, 2005 masque.... Serological assays: FAMA (Fluorescent Antibody Membrane Assay) Williams, et al. J Infect Dis,1974,130: 669-672. Antigen: unfixed VZV-infected cells Output: Fluorescence Only serological test known to correlate protection from infection with a specific titer of antibodies Highly sensitive, probably due to the use of unfixed cells in which conformational structures of VZV proteins are preserved  « » in sero-  « Gold Standard » in sero- epidemiological surveys when epidemiological surveys when low levels of antibodies low levels of antibodies By courtesy of Dr. A. Gershon

11. Rome, Italy - March 18-19, 2005 masque.... BUT Not widely available (one kit commercialized??) Labor-intensive: cell-culture, live virus Requires experience in reading and interpreting the results Subjectivity of the output Serological assays: FAMA (Fluorescent Antibody Membrane Assay) Williams, et al. J Infect Dis,1974,130: 669-672.

12. Rome, Italy - March 18-19, 2005 masque.... Serological assays: IFAT (Indirect Fluorescent Antibody Test) Sauerbrei A, et al. J. Virol. Methods, 2004;119:25-30 Antigen: A549 (human lung carcinoma cells) + 0.0001 m.o.i, removed mechanically 7- 10dpi (10% CPE) and fixed with acetone 1h -20°C (stable 6 months) Output: fluorescence Sera serially diluted (first dilution 1:5) (18h at RT + 3h 37°C)

13. Rome, Italy - March 18-19, 2005 masque.... Comparison between FAMA and IFAT: Antibody Titer: 5x higher with IFAT, for low titer sera 8x higher with IFAT, for high titer sera Serological assays: IFAT (Indirect Fluorescent Antibody Test) Reproduced from Sauerbrei A et al. J Virol Methods 2004; 119: 25–30 with permission from Elsevier (http://www.sciencedirect.com/science/journal/01660934)

14. Rome, Italy - March 18-19, 2005 masque.... Validation of FAMA and IFAT using the British Standard for VZV antibodies (National Institute for Biological Standards and Control, Hertfordshire, UK): 4 IU anti- VZV IgG/ml –FAMA: 250mIU/ml (highest dilution showing a pos. Result: 1/16) –IFAT: 50mUI/ml (highest dilution showing a pos. Result: 1/80)  Both tests are sensitive BUT Subjective output Not commercialized Serological assays: IFAT (Indirect Fluorescent Antibody Test) Sauerbrei A, et al. J. Virol. Methods, 2004;119:25-30

15. Rome, Italy - March 18-19, 2005 masque.... Serological assays: LA (Latex Agglutination) Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9. Antigen: VZV antigen coated on latex particles Output: obvious clumping of latex particles Subjectivity Easy and rapid to perform (15min) Commercialized Comparable to FAMA: Only 2% FAMA neg/LA pos (not true neg?)

16. Rome, Italy - March 18-19, 2005 masque.... FAMALA Healthy children0% Sera prior to or on the day of onset of varicella 0% Convalescent-phase (2 sera from the same individual) 100%96% Post-vaccine77%61% Serological assays: LA (Latex Agglutination) Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.

17. Rome, Italy - March 18-19, 2005 masque.... Serological assays: LA (Latex Agglutination) Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527- 9. Percentage of positive samples FAMA assayLAELISA Healthy adults695236 Healthy children937661 Leukemic children725748 Total776147 P vs FAMA–0.0010.0001 P vs LA––0.01

18. Rome, Italy - March 18-19, 2005 masque.... FAMA/LA: Serological assays: LA (Latex Agglutination) Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9. Number of FAMA results LA result PositiveNegativeTotal Positive37946425 Negative11442453 Total390488878 48% 44% Sensitivity: 92% agreement Specificity: 93% agreement 52% 56% Comparison of VZV antibody determinations by LA and FAMA assay The rate of positivity is not significantly different between these assays (P>0.05)

19. Rome, Italy - March 18-19, 2005 masque.... Sensitive Specific (no reactivity with other herpesviruses) Reproducible  Comparable to FAMA BUT False negative (Prosone): need to retest the samples that appear neg at the 1:2 dilution Serological assays: LA (Latex Agglutination) Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.

20. Rome, Italy - March 18-19, 2005 masque.... ANTIGENS Commer- cialized DISADVANTAGESADVANTAGES ELISA (Enzyme linked immunosorbent assay) Extract from VZV- infected cells + - Lack of sensitivity - Lack of specificity -Automated - Objectivity of the output - Useful for numerous samples analysis gpELISA VZV purified Glycoproteins - oversensitive?See ELISA LA (Latex Agglutination) VZV proteins coated on latex particles + - Subjectivity of the output - false- negative results in Prosone - Rapid - Useful for numerous samples FAMA (Fluorescent Antibodies to Membrane Antigens) Live, unfixed VZV- infected cells +? - Subjectivity of the output: need of experience for reading and interpretation - cannot be automated -Sensitive -Specific -Good correlation with protection IFAT (Immuno- Fluorescent Antibody Test) Acetone-fixed VZV- infected A549 cells - - Subjectivity of the output: need of experience for reading and interpretation - cannot be automated -Sensitive -Specific -Good correlation with protection

21. Rome, Italy - March 18-19, 2005 masque.... Serological assays: conclusions Need for harmonization of the tests used all over Europe to be able to compare results. However, no serologic test is perfect. Which assay ??? FAMA = « gold standard » but requires a lot of experience, and difficult to use for large-scale studies IFAT? LA? ELISA? First screening with one test and retesting with a second assay? Comparison using the reference serum as control?