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NetMHCIIpan - 4.3

Pan-specific binding of peptides to MHC class II molecules of known sequence

The NetMHCIIpan-4.3 server predicts peptide binding to HLA class II molecules using Artificial Neural Networks (ANNs). It is trained on an extensive dataset of over 650,000 measurements of Binding Affinity (BA) and Eluted Ligand mass spectrometry (EL), covering the three human MHC class II isotypes HLA-DR, HLA-DQ, HLA-DP, as well as mouse (H-2) and bovine (BoLA-DRB3) molecules.

The network can predict for any HLA class II molecule of known sequence, which the user can specify as FASTA format, and predictions can be made for peptides of any length.

The output of the model is a prediction score for the likelihood of a peptide to be naturally presented by an MHC-II receptor of choice. The output also includes a %rank score, which normalizes the prediction score by comparing to predictions of a set of random peptides. Optionally, the model also outputs BA prediction and %rank scores.

New in version 4.3: The method is trained on an extended EL dataset including new data for HLA-DP, HLA-DR and BoLA-DRB3. Further, the method allows for prediction of inverted peptide binders.

Refer to the instructions page for more details.

The project is a collaboration between DTU-Bioinformatics, and LIAI.

View the version history of this server

Updated March 18 2024: A bug related to specified peptide lengths longer than the input FASTA sequence(s) has been fixed.

SUBMISSION

Hover the mouse cursor over the symbol for a short description of the options

INPUT TYPE:

Paste a single sequence or several sequences in FASTA format into the field below:

... or upload a file in FASTA format directly from your local disk:

... or load some sample data:


PEPTIDE LENGTH (specify variable length as a comma separated list):  


Use context encoding


SELECT Molecules:



Select Allele(s) (max. 15 per submission)



... or type a list of molecules names separated by commas without spaces (max 15 per submission)

For the list of available molecule names click here

Alternatively, upload full length Alpha and Beta chain protein sequences:


ADDITIONAL CONFIGURATION:

Threshold for strong binder (% Rank)  
Threshold for weak binder (% Rank)  

Include BA predictions

Allow peptide inversion for other loci than HLA-DP 

Turn on filtering options 

Print only the strongest binding core 
Sort output by prediction score 

Save predictions to xls file 

Restrictions:
At most 5000 sequences per submission; each sequence not more than 20,000 amino acids and not less than 9 amino acids. Max 15 MHC alleles per submission.

Confidentiality:
The sequences are kept confidential and will be deleted after processing.

CITATIONS

For publication of results, please cite:

    Accurate prediction of HLA class II antigen presentation across all loci using tailored data acquisition and refined machine learning
    Jonas B. Nilsson, Saghar Kaabinejadian, Hooman Yari, Michel G. D. Kester, Peter van Balen, William H. Hildebrand and Morten Nielsen
    Science Advances, 24 Nov 2023. https://www.science.org/doi/10.1126/sciadv.adj6367

PORTABLE VERSION

NetMHCIIpan 4.3 is available as a stand-alone software package, with the same functionality as the service above. Ready-to-ship packages exist for Linux and macOS. There is a download page for academic users; other users are requested to contact Health Tech Software Package Manager at health-software@dtu.dk.

Instructions

INPUT DATA

In this section, the user must define the input for the prediction server following these steps:

1) Specify the desired type of input data (FASTA or PEPTIDE ) using the drop down menu.

2) Provide the input data by means of pasting the data into the blank field, uploading it using the "Choose File" button or by loading sample data using the "Load Data" button. All the input sequences must be in one-letter amino acid code. The alphabet is as follows (case sensitive):

A C D E F G H I K L M N P Q R S T V W Y and X (unknown)

Any other symbol will be converted to X before processing. At most 5000 sequences are allowed per submission; each sequence must be not more than 20,000 amino acids long and not less than 9 amino acids long.

3) If FASTA was selected as input type, the user must select the peptide length(s) the prediction server is going to work with. NetMHCIIpan-4.3 will "chop" the input FASTA sequence in overlapping peptides of the provided length and will predict binding against all of them. By default input proteins are digested into 15-mer peptides. Note that, if PEPTIDE was selected as input type, this step is unnecessary and thus the peptide length selector will directly not appear in the interface.

4) Context encoding informs the network of the proteolytic context the ligand. Context is automatically generated from the source protein if the user selects FASTA format. Briefly, context is made up of 12 amino acids: 3 amino acids upstream of the ligand, 3 first amino acids at the ligand N-terminus, 3 last amino acids at the ligand C-terminus and 3 amino acids downstream the ligand(in the source protein), all concatenated together. If the input type is PEPTIDE , the user must specify the ligand context(see PEPTIDECONT ).

Input




MHC SELECTION

In this section, the user must define which MHC molecule(s) the input data is going to be predicted against:

1) Here the user can select from a list of MHC molecules by first selecting the group and clicking MHCs in the list. Here, MS-COVERED refers to molecules properly covered by the NetMHCIIpan-4.3 training data.

2) The user can also type the molecule names. Both the ALPHA and BETA chains must be typed (please consult List of MHC molecule names.) Note that molecules selected from step 1 populate this bar.

3) If the molecule of interest is not provided in the lists, the user can input ALPHA and BETA sequences in fasta format. With this option, rank score predictions are not available.

MHCSelection

ADDITIONAL CONFIGURATION

In this section, the user may define additional parameters to further customize the run:

1) Specify thresholds for strong and weak binders. They are expressed in terms of %Rank, that is percentile of the predicted binding affinity compared to the distribution of affinities calculated on set of random natural peptides. The peptide will be identified as a strong binder if it is found among the top x% predicted peptides, where x% is the specified threshold for strong binders (by default 2%). The peptide will be identified as a weak binder if the % Rank is above the threshold of the strong binders but below the specified threshold for the weak binders (by default 10%).

2) Tick this option to include also Binding Affinity predictions together with Eluted Ligand likelihood.

3) Enable this option to allow prediction of peptide inversion for all the selected MHC-II molecules. By default, inversion is only considered for HLA-DP as only this locus has been shown to allow widespread peptide inversion. Nonetheless, it may be of interest for the user to allow peptide inversion for all the input molecules, and in this case this option should be enabled. Note that predictions will take longer to run with this option enabled.

4) Tick this option to output only peptides with a % Rank score below a specified threshold. Useful for large submissions.

5) Tick this box to output only the strongest binding core.

6) Tick this box to have the output sorted by descending prediction score.

7) Enable this option to export the prediction output to .XLS format (readable for most spreadsheet softwares, like Microsoft Excel).


MHCSelection

SUBMISSION

After the user has finished the "INPUT DATA", "MHC SELECTION" and "ADDITIONAL CONFIGURATION" steps, the submission can now be done. To do so, the user can click on "Submit" to submit the job to the processing server, or click on "Clear fields" to clear the page and start over.

The status of your job (either 'queued' or 'running') will be displayed and constantly updated until it terminates and the server output appears in the browser window.

After the server has finished running the corresponding predictions, an output page will be delivered to the user. A description of the output format can be found at outpur format

At any time during the wait you may enter your e-mail address and simply leave the window. Your job will continue; when it terminates you will be notified by e-mail with a URL to your results. They will be stored on the server for 24 hours.
OutputFormat

Output format

EXAMPLE OUTPUT

For the following FASTA input example:

>Q5QFB9
MRYGFVRKKHRGLFLTTVAALPIWNPISEFVKWYKSHKLSQHCIRICGHLCQKHLDMFLSVIGQRWPIDVFSSVFDHQVSAIGSDIIWWFLKLFLVSFFFFF

With parameters:

Peptide length: 15
Allele: HLA-DPA10202-DPB11901
Sort by prediction score: On

NetMHCIIpan-4.3 will return the following output (showing the top 10 predicted peptides): 

# NetMHCIIpan version 4.3d

# Input is in FASTA format

# Peptide length 15

# Prediction Mode: EL

# Threshold for Strong binding peptides (%Rank)  1.00%
# Threshold for Weak binding peptides (%Rank)    5.00%

# HLA-DPA10202-DPB11901 : Distance to training data  0.000 (using nearest neighbor HLA-DPA10202-DPB11901)

# Allele: HLA-DPA10202-DPB11901
--------------------------------------------------------------------------------------------------------------------------------------------
 Pos                     MHC              Peptide   Of        Core  Core_Rel Inverted        Identity      Score_EL %Rank_EL  Exp_Bind  BindLevel
--------------------------------------------------------------------------------------------------------------------------------------------
  25   HLA-DPA10202-DPB11901      NPISEFVKWYKSHKL    4   KYWKVFESI     0.980        1          Q5QFB9      0.600852     0.84        NA   <= SB
  24   HLA-DPA10202-DPB11901      WNPISEFVKWYKSHK    3   KYWKVFESI     0.970        1          Q5QFB9      0.567158     1.02        NA   <= WB
  26   HLA-DPA10202-DPB11901      PISEFVKWYKSHKLS    5   KYWKVFESI     0.930        1          Q5QFB9      0.459227     1.76        NA   <= WB
  23   HLA-DPA10202-DPB11901      IWNPISEFVKWYKSH    2   KYWKVFESI     0.880        1          Q5QFB9      0.319686     3.48        NA   <= WB
  27   HLA-DPA10202-DPB11901      ISEFVKWYKSHKLSQ    3   KHSKYWKVF     0.420        1          Q5QFB9      0.239845     5.02        NA
  28   HLA-DPA10202-DPB11901      SEFVKWYKSHKLSQH    4   KWYKSHKLS     0.680        0          Q5QFB9      0.216487     5.63        NA
   6   HLA-DPA10202-DPB11901      VRKKHRGLFLTTVAA    3   KHRGLFLTT     0.980        0          Q5QFB9      0.161751     7.49        NA
  22   HLA-DPA10202-DPB11901      PIWNPISEFVKWYKS    1   KYWKVFESI     0.590        1          Q5QFB9      0.147204     8.15        NA
  29   HLA-DPA10202-DPB11901      EFVKWYKSHKLSQHC    5   YKSHKLSQH     0.440        0          Q5QFB9      0.128036     9.16        NA
   5   HLA-DPA10202-DPB11901      FVRKKHRGLFLTTVA    4   KHRGLFLTT     0.850        0          Q5QFB9      0.113368    10.07        NA

DESCRIPTION


The prediction output for each molecule consists of the following columns:

  • Pos Residue number (starting from 0)

  • MHC MHC molecule name

  • Peptide Amino acid sequence

  • Of Starting position offset of the optimal binding core (starting from 0)

  • Core Binding core register

  • Core_Rel Reliability of the binding core, expressed as the fraction of networks in the ensemble selecting the optimal core

  • Inverted Whether the peptide binds inverted to the given MHC molecule (1: inverted, 0: forward)

  • Identity Annotation of the input sequence, if specified

  • Score_EL Eluted ligand prediction score

  • %Rank_EL Percentile rank of eluted ligand prediction score

  • Exp_bind If the input was given in PEPTIDE format with an annotated affinity value (mainly for benchmarking purposes).

  • Score_BA Predicted binding affinity in log-scale (printed only if binding affinity predictions were selected)

  • Affinity(nM) Predicted binding affinity in nanomolar IC50 (printed only if binding affinity predictions were selected)

  • %Rank_BA % Rank of predicted affinity compared to a set of 100.000 random natural peptides. This measure is not affected by inherent bias of certain molecules towards higher or lower mean predicted affinities (printed only if binding affinity predictions were selected)

  • BindLevel (SB: strong binder, WB: weak binder). The peptide will be identified as a strong binder if the % Rank is below the specified threshold for the strong binders. The peptide will be identified as a weak binder if the % Rank is above the threshold of the strong binders but below the specified threshold for the weak binders.

    • Article abstracts

    Accurate prediction of HLA class II antigen presentation across all loci using tailored data acquisition and refined machine learning

    Jonas B. Nilsson, Saghar Kaabinejadian, Hooman Yari, Michel G. D. Kester, Peter van Balen, William H. Hildebrand and Morten Nielsen


    Science Advances, 24 Nov 2023. https://www.science.org/doi/10.1126/sciadv.adj6367
    Accurate prediction of antigen presentation by human leukocyte antigen (HLA) class II molecules is crucial for rational development of immunotherapies and vaccines targeting CD4⁺ T cell activation. So far, most prediction methods for HLA class II antigen presentation have focused on HLA-DR because of limited availability of immu-nopeptidomics data for HLA-DQ and HLA-DP while not taking into account alternative peptide binding modes. We present an update to the NetMHCIIpan prediction method, which closes the performance gap between all three HLA class II loci. We accomplish this by first integrating large immunopeptidomics datasets describing the HLA class II specificity space across all loci using a refined machine learning framework that accommodates inverted peptide binders. Next, we apply targeted immunopeptidomics assays to generate data that covers additional HLA-DP specificities. The final method, NetMHCIIpan-4.3, achieves high accuracy and molecular coverage across all HLA class II allotypes.

    Supplementary material

    Training data

    Here, you will find the data set used for training of NetMHCIIpan-4.3.

    NetMHCIIpan_train.tar.gz

    Download the file and untar the content using 

    cat NetMHCIIpan_train.tar.gz | tar xvf -

    This will create the directory called NetMHCIIpan_train. In this directory you will find 12 files. 10 files (c00?_ba, c00?_el) with partitions with binding affinity (ba) or eluted ligand data (el). The format for each file is (here shown for an el file) 

    AAAAMAEQESARN 1 Saghar_9061_DR MAAAAAARNGGR
AAAAVQGGRSGG 1 Saghar_9090_DR MAAAAVSGGSGG
AAALEAMKDYTKAM 1 Saghar_9013_DR TRKAAAKAMDVY
AAALEAMKDYTKAMD 1 Saghar_9013_DR TRKAAAAMDVYQ
AAEFIQQFNNQAFS 1 Saghar_9090_DR DKMAAEAFSVGQ
AAEFIQQFNNQAFSVG 1 Saghar_9090_DR DKMAAESVGQQL
AAFPFLAYSGIPAVS 1 Saghar_9013_DR LDNAAFAVSFCF
AAGQFFPEAAQVAYQ 1 Saghar_9090_DR DDDAAGAYQMWE
AAGVTDGNEVAKA 1 Saghar_9061_DR VRGAAGAKAQQA
AAIRKKLVIVGD 1 Saghar_9013_DR MAAIRKVGDGAC
    where the different columns are peptide, target value, MHC_molecule/cell-line, and context. In cases where the 3rd columns is a cell-line ID, the MHC molecules expressed in the cell-line are listed in the allelelist file.

    The allelelist file contains the information about alleles expressed in each cell line data set, and pseudosequence.2023.dat the MHC pseudo sequence for each MHC molecule.

    Benchmark data

    You can download the benchmark dataset used in the NetMHCIIpan-4.3 publication here:
    NetMHCIIpan_eval.fa Each entry has the following format: 
    >ID Epitope HLA
Sequence
    where ID is the Uniprot identifier, Epitope is the epitope, HLA is the HLA molecule bound by the epitope, and Sequence is the source protein sequence in which the epitope is derived.

    Version history

    Please click on the version number to activate the corresponding server.

    4.3 The current version (online since July 2023). New in this version:
    • NetMHCIIpan-4.3 is trained on an extended dataset of MHC eluted ligands, incorporating new data for HLA-DP, HLA-DR and BoLA-II. Furthermore, an update to the NNAlign_MA machine learning framework allows for prediction of inverted peptide binders.
    Main publication:
    • Accurate prediction of HLA class II antigen presentation across all loci using tailored data acquisition and refined machine learning
      Jonas B. Nilsson, Saghar Kaabinejadian, Hooman Yari, Michel G. D. Kester, Peter van Balen, William H. Hildebrand and Morten Nielsen
      Science Advances, 24 Nov 2023. https://www.science.org/doi/10.1126/sciadv.adj6367
    4.2 (online since September 2022). New in this version:
    • NetMHCIIpan-4.2 is trained on an extensive dataset of both eluted ligand (EL) and binding affinity (BA) data, including new novel EL data for 14 HLA-DQ molecules. Further, a 'distance to training data' metric is printed for each selected molecule in the same way as NetMHCpan-4.1, indicating how reliable the predictions are.
    Main publication:
    • Machine learning reveals limited contribution of trans-only encoded variants to the HLA-DQ immunopeptidome
      Jonas Birkelund Nilsson, Saghar Kaabinejadian, Hooman Yari, Bjoern Peters, Carolina Barra, Loren Gragert, William Hildebrand and Morten Nielsen
      Communications Biology, 21 April 2023. https://doi.org/10.1038/s42003-023-04749-7
    4.1 (online since Sept 2021). New in this version:
    • The method is trained on a extented set of EL data compared to version 4.0, and novel and correct BA for HLA-DQA1*04:01-DQB1*04:02 are included. Further is DRB3, 3 and 5 allele information predicted from DRB1 based on linkage disequilibrium with DRB1 when absent from typing data.
    Main publication:

    • Accurate MHC Motif Deconvolution of immunopeptidomics data reveals high relevant contribution of DRB3, 4 and 5 to the total DR Immunopeptidome
      Saghar Kaabinejadian, Carolina Barra, Bruno Alvarez, Hooman Yari, William Hildebrand, Morten Nielsen
      Frontiers in Immunology 26 January 2022. Sec. Antigen Presenting Cell Biology, DOI: 10.3389/fimmu.2022.835454
    4.0 (online since April 2020). New in this version:
    • The two output neuron architechture introduced in NetMHCpan-4.0 permits the inclusion of EL data, and the new training algorithm NNAlign_MA extends training data to ligands of ambiguous allele assignments. The model also, optionally, encodes ligand context.
    Main publication:

    • Improved prediction of MHC II antigen presentation through integration and motif deconvolution of mass spectrometry MHC eluted ligand data.
      Reynisson B, Barra C, Kaabinejadian S, Hildebrand WH, Peters B, Nielsen M
      J Proteome Res 2020 Apr 30. doi: 10.1021/acs.jproteome.9b00874.
      PubMed: 32308001
    3.2 (online since January 2018). New in this version:
    • Method retrained on an extensive dataset of over 100,000 datapoints, covering 36 HLA-DR, 27 HLA-DQ, 9 HLA-DP, and 8 mouse MHC-II molecules.
    Main publication:

    • Improved methods for predicting peptide binding affinity to MHC class II molecules.
      Jensen KK, Andreatta M, Marcatili P, Buus S, Greenbaum JA, Yan Z, Sette A, Peters B, Nielsen M.
      Immunology. 2018 Jan 6. doi: 10.1111/imm.12889.
      PubMed: 29315598
    3.1 (online since December 2014). New in this version:
    • Improved binding core identification by realigning individual networks in the ensemble.
    • Introduced a reliability measure on the predicted binding core (Core_Rel column).
    • Graphical representation of the binding core register and of possible multiple cores.
    Main publication:

    • Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification
      Andreatta M, Karosiene E, Rasmussen M, Stryhn A, Buus S, and Nielsen M
      Immunogenetics (2015)
      PubMed: 26416257
    3.0 (online since June 2013). New in this version:
    • The user can make predictions for all DR, DP and DQ molecules with known protein sequence. Likewise can the user upload full length MHC class II alpha and beta chain and have the server predict MHC restricted peptides from any given protein of interest
    2.1 (online since 6 June 2011). New in this version:
    • User can upload full length MHC class II beta chain and have the server predict MHC restricted peptides from any given protein of interest.
    2.0 (online since 17 Nov 2010). New in this version:
    • New concurent algorithm used to train the network.
    1.1 (online since 15 April 2010). New in this version:
    • %-rank measure include for each prediction value. The %-rank score give the rank of the prediction score to a distribution of prediction scores from 200.000 natural random 15mer peptides.
    1.0 Original version (online version until April 15 2010):

    Main publication:

    • Quantitative predictions of peptide binding to any HLA-DR molecule of known sequence: NetMHCIIpan.
      Nielsen M, et al. (2008) PLoS Comput Biol. Jul 4;4(7):e1000107. View the abstract, the full text version at PLoS Compu: Full text.

    Software Downloads


    GETTING HELP

    If you need help regarding technical issues (e.g. errors or missing results) contact Technical Support. Please include the name of the service and version (e.g. NetPhos-4.0). If the error occurs after the job has started running, please include the JOB ID (the long code that you see while the job is running).

    If you have scientific questions (e.g. how the method works or how to interpret results), contact Correspondence.

    Correspondence: Technical Support: