Epitope analysis with

MatchIt software

Ivan Balazs, Ph.D.

HLA Epitopes in Clinical TransplantationPittsburgh, PA

June 15-16, 2011

Epitope Analysis with single antigens

• Why do it:– Rationalize/explain serum reactivity patterns– Distinguish between Donor vs de novo antibodies– Facilitate assignment of permissible and non-permissible

antigens

• Information needed: – Patient HLA typing (4 digit res.)– Donor HLA typing (4 digit res.)– Pre-Transplant Ab sensitization events– Post-Transplant Ab results– List of functional epitopes

Epitope Analysis with single antigens.

Confounding events (life is not easy!!!):• Unknown number and source of sensitizing event

– Transfusions (blood or components)– Pregnancies & aborted pregnancies

• Sensitization to multiple Ag(s) and similar or very different levels of Abs

• There may be epitopes not accounted for (e.g. effect of internal aa changes on Ag conformation and antibody affinity)

Unfortunately not every case lends itself to this type of analysis.

Confounding events (continued)

Sample analysis requires time and money.• Mixture of Ab and presence of multiple IgG

subtypes (Adsorption/elution with single Ags; Subtype specific Ab)

• Interference by IgM (inactivate or eliminate)• High titer serum (signal detector saturation)

– Repeat assay at 1:4, 1:16 dilution to bring within range

Therefore, not every case can be analyzed

MatchIt Softwareantibody analysis

• MatchIt software for Ab was designed as a LIMS system for the analysis of serological results obtained with Lifecodes products

• MatchIt software can also be used for epitope assignment

• HLAMatchmaker software for epitope sharing, permissible/non-permissible Ag assignments

MatchIt software for antibody analysisEpitope assignment

• It utilizes the list of epitopes proposed by Duquesnoy in HLAMatchmaker and publications:– M. Marrari & R. J. Duquesnoy. Correlations between Terasaki’s HLA class II

epitopes and HLAMatchmaker-defined eplets on HLA-DR and -DQ antigens. Tissue Antigens (2009): 70: 134

– R. J. Duquesnoy & M. Marrari. Correlations between Terasaki’s HLA class I epitopes and HLAMatchmaker-defined eplets on HLA-A, -B and -C antigens. Tissue Antigens (2009): 70: 117

• Customizable list of epitopes and specificities i.e. import/export list)

• Automated assignment of epitopes by tail analysis.• Possibility to perform manual assignments

Epitope distribution in Class I Ag

Automated Tail Analysis of epitopes

Maual epitope Analysis

Donor HLA Type A2,A11,B7,B40

Examples of Class I epitopes

Donor: A*24:02

Examples of Class I epitopes

MM Donor: A*02:05

Examples of Class I epitopes

MM Donor: A*35:01

Examples of Class II epitopes

MM Donor: DQA*03:02/B*03:03

Examples of Class II epitopes

MM Donor: DQA*05:01/B*03:01

Examples of Class II epitopes

MM Donor: DRB3*01:01 DQB*03:01

There is still a lot to be learned

• To help interpret the patterns of antibody reactivity we need more experimental results for:– Empirical confirmation of immunogenic epitopes

(modeling is just the start)– Estimating relative frequency/immunogenicity of

different epitopes (not all mismatched Ag are equally immunogenic) (F. Claas et al.)

– What is the effect of the other patient’s Ags on the immunogenicity of the mismatched antigens (shared CDR?)

16th International HLA and

Immunogenetics Workshop

Liverpool, UK28th May - 3rd June 2012

Project: Development of an HLA Epitope Database

Rene Duquesnoy

University of Pittsburgh

16th International HLA and Immunogenetics Workshop

• Committee: Rene Duquesnoy (Pittsburgh, PA) Chair, Domenico Adorno (L’Aquila, Italy), Ivan Balazs (Stamford, CT, USA), Frans Claas (Leiden, The Netherlands), Nadim El-Awar (Los Angeles, CA, USA), Marcelo Fernandez-Vina (Houston, TX, USA), Nils Lachmann (Berlin, Germany), Derek Middleton (Liverpool, UK), Arend Mulder (Leiden, The Netherlands), Craig Taylor (Cambridge, UK), Robert Vaughan (London, UK) and Cristina von Glehn (Curitiba, Brazil)

• This committee has the following tasks:• Determine criteria for the experimental verification of epitopes reacting

with informative antibodies.  Address the question of clinically relevant versus irrelevant epitopes.

• Develop a notation system for Class I and Class II epitope repertoires• Verify experimentally defined epitopes submitted by workshop

participants• Develop a website based HLA epitope database• Please note that this project addresses only antibody-reactive epitopes, not

cellular epitopes defined by T-cells.

Acknowledgements

Gen-Probe Transplant Diagnostics, Inc.

Bryan RayAlexandra StormJustin Mostecki

Jayasree HariharanLioudmila Zaslavskaia