Epitope discovery inarenaviruses

Outline

• Introduction to arenaviruses• How to find the data? • How to predict epitopes• What sequences to include?• How to estimate conservation?

• Prediction of epitopes in Junin virus

Why focus on arena virus?

How to get data!

NCBI has it all!(http://www.ncbi.nlm.nih.gov/)

NCBI has it all!(http://www.ncbi.nlm.nih.gov/)

NCBI has it all!(http://www.ncbi.nlm.nih.gov/)

How to predict?

Phylogeny Hanta virus

Puuma Virus

Dobrava Virus

Hantaan Virus

Seoul Virus

Thottapalayam

AF482715

Tula Virus

Hanta VirusLodz-2

Muleshou Hanta Virus

Laguna Virus

Toparafou Hanta Virus

Hanta Segment S conservation

• Focus on Segment S protein in Hanta

• 7 full length segment S sequences in Swissprot

• Pairwise %id > 95%• What is the overlap in 9mers?

Segment D sequences

Oran Virus

Hanta Virus

Hanta Virus

Andes Virus

Pergamino Virus

Maciel virus

Bermejo Virus

Lechiguanas Virus

Hanta Segment S conservation

Epitope identification

•AF482715 contains 420 unique 9mers•AF482712, AF482711, AF291702, AF482717, AF482716, AF482715, AF482714, AF482713, AF324902 contain 734 unique peptides•Only 243 9meric peptides are conserved among all 9 sequences

Viral sequence conservation

No two viral strains will “host” the same set of CTL eitopes

HLA supertypes

Epitope predictions

• NetCTL• Include all 12 supertypes• Combined score > 1.0• Identify epitopes that are most conserved in the different proteins in the Junin/Hanta sequences

• Verify that the majority of the selected epitopes are present in the Argentina viral strains•Validation will be carried out in Argentina

Promiscuous epitopes

Supertype Peptide MHC C9 TAP netCTL A26 YIKGGNASF 1.6511 0.9755 2.6890 1.9319 B62 YIKGGNASF 1.2262 0.9755 2.6890 1.5070 B27 FQFFVFLAL 0.9887 0.9689 1.0950 1.1887 B39 FQFFVFLAL 1.7054 0.9689 1.0950 1.9054 B7 LPRRSLKAF 1.5699 0.8476 2.2340 1.8088 B8 LPRRSLKAF 1.8902 0.8476 2.2340 2.1290 B39 LQEALNIAL 1.1432 0.2856 0.7850 1.2253 B62 LQEALNIAL 1.0011 0.2856 0.7850 1.0832 B7 IPTFLQEAL 1.3419 0.9521 0.4700 1.5083 B8 IPTFLQEAL 0.8476 0.9521 0.4700 1.0139 A1 YLSPFKLTY 2.5764 0.9788 2.9670 2.8716 B62 YLSPFKLTY 1.0820 0.9788 2.9670 1.3771 A1 ALTSLGLLY 1.8120 0.8517 3.0030 2.0899 A3 ALTSLGLLY 1.1383 0.8517 3.0030 1.4162

Epitope conservation

• 23 of the 23 select L epitopes are present in the 5 L protein sequences• 6 of the 7 selected Z epitopes are present in the 5 Z proteins sequences. 1 epitope is present in 4 Z protein sequence• 26 of the 28 selected GPG epitopes are present in all 9 complete GPC sequences. 1 is present in 4 of the 9. 1 epitope is absent in the complete GPC sequences.

• 1 of the selected GPC fragment epitopes is present in only 5 GPC fragment sequences. 26 of the selected 27 NP epitopes are present in the 12 complete NP sequences. One is present in only 3.

• This latter epitopes is also only present in 7 of the 54 NP fragment sequences.

Overlap with the Argentinean strain• 26 of the 27 epitopes selected from the NP and NP fragment proteins are found in the ABB80080 sequence. (DQ272266 genbank segment S).• 27 of the 28 epitopes selected from the GPC and GPC fragments are found in the NP_899218 sequence.• 7 of the 7 epitopes selected from the Z protein are found in the AAV68494 sequence (AY819707 genbank segment L). • 23 of the 23 epitopes selected form the L protein are found in the AAV68495 sequence (AY819707 genbank segment L).

Whats next

• Obtain blood samples from Junin/Hanta infected patients• Elispot immunoassays to validate immunogenecity of predicted epitopes