The quest for the needle (antigen) in the haystack (pathogen):

Immunoinformatics to the rescue

Nicholas Svitek, Lucilla Steinaa, Rosemary Saya, Elias Awino, Morten Nielsen, Søren Buus, Vishvanath Nene

ILRI BioSciences Day, Nairobi, 27 November 2013

Killing

Immune Response Elicited during Theileriaparva Infection: Cellular Immunology 101

T. parva infected white blood cells

The butler (the MHC class I molecule)is serving T. parva peptides to the “Killer T cell”

T CD8+= CTL(killers of

infected cells)

T. parva

T. parvapeptides

The butler

CTL need to find infected cells through a lock-key mechanism

The Challenge: Many Locks & Keys Are Available and these need to be Tracked Down

T. parva T. parva

T. parvapeptides

T. parvapeptides

The butler The butler

The Challenge: Searching for an Antigen is like Searching for a Needle in a Haystack

The Problem: using Conventional Methods for Ag ID are Expensive & Time Consuming

+

Mugugacocktail

Oxytetracycline

Infection & Treatment Method (ITM)

Restricting BoLA

class I molecule

T. parva

antigen CTL Epitope

1) 6*01301 Tp1 214VGYPKVKEEML224

2) 6*04101 Tp2 27SHEELKKLGML37

3) 2*01201 Tp2 49KSSHGMGKVGK59

4) BoLA-T2c Tp2 96FAQSLVCVL104

5) BoLA-T5 Tp5 87SKADVIAKY95

6) 3*00101 Tp8 379CGAELNHFL387

7) 1*02301 Tp9 199AKFPGMKKSK208

Induction of lifelong CTL response

Reverse Immunology as a Method to Speed Up Antigen Identification

Whole Genome Sequence from T. parva

In silico antigen predictions In vitro characterization of predicted antigens

Selection of immunodominant peptides

VaccinateNaïve cattle

Challenge with T. parva

Computer algorithmstrained on biological data

Immunoinformatics

Computer algorithmstrained on biological data(NetMHCpan)

T. parva genome

Prediction of parasite peptides that binds to

bovine MHC class I molecules

Reverse Immunology : the NetMHCpan Platform

Results 1: The True CTL Epitope that Binds to BoLA-6*04101 is Tp229-37

Antigen Binder

Tp2 27SHEELKKLGML37 6*04101 NO (FP 0.058)

Tp2 29EELKKLGML37 6*04101 YES (FP 0.002)

NetMHCpan:

Binding assay:

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Tp2.29-37 (Alt)

Tp2.27-37

Tp229-37

Tp227-37

O.D

. (@

450 n

m)

[Peptide]

Variants:SHEELKKLGML

Var1 SDEELKKLGMLVar2 SDDELDTLGMLVar3 SNEELKKLGMVVar4 TNEELKKLGMV

CD8

http://flow.csc.mrc.ac.uk/?page_id=852

CD8+

CD8+/Tetramer +

β2m

Recombinant MHC Production Tetramerization

Incubation for

24-48 hours

Using peptide-MHC Class I Tetramers to Confirm CTL Specificity towards Predicted Epitope

Results 2: Identification of Tp1 p-MHC Tetramer Positive Cells in Ex Vivo Assays

Day 8 p.i.

Day 15-17 p.i.

Animal 1 Animal 2 Animal 3

Tp

1+

(P

E)

CD8+

(PerCP)

Conclusion

• Immunoinformatic (NetMHCpan) can potentially be used to identify new CTL epitopes.

• p-MHC class I tetramers & NetMHCpan can be used for correct epitope mapping.

- Combining NetMHCpan with other prediction parameters; in collaboration with Dr. Morten Nielsen:

• Amino acid composition

• Protein functional category

• Subcellular localization

- T. parva comparative genomics to identify proteins that are under positive selection [potential targets of the immune response] ; in collaboration with Dr. Joana Silva.- Use of immunoinformatics for ASF virus antigen identification (Dr. Richard Bishop).

Where to from now?

Acknowledgements

Søren Buus,tetramers

Morten Nielsen,NetMHCpan

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