Medical Microbiology and Immunology

, Volume 208, Issue 2, pp 227–238 | Cite as

Conserved peptide vaccine candidates containing multiple Ebola nucleoprotein epitopes display interactions with diverse HLA molecules

  • Sahil Jain
  • Manoj BaranwalEmail author
Original Investigation


Immunoinformatics has come by leaps and bounds to finding potent vaccine candidates against various pathogens. In the current study, a combination of different T (CD4+ and CD8+) and B cell epitope prediction tools was applied to find peptides containing multiple epitopes against Ebola nucleoprotein (NP) and the presentation of peptides to human leukocyte antigen (HLA) molecules was analyzed by prediction, docking and population coverage tools. Further, potential peptides were analyzed by ELISA for peptide induced IFN-γ secretion in peripheral blood mononuclear cells isolated from healthy volunteers. Six peptides were obtained after merging the overlapping multiple HLA I (CD8+) and II (CD4+) restricted T cell epitopes as well as B cell epitopes and eliminating the peptides liable to generate autoimmune and allergic response. All peptides displayed 100% conservancy in Zaire ebolavirus. In other Ebola virus species (Sudan, Bundibugyo and Taï forest) and Filoviridae members (Lloviuvirus and Margburgvirus), some peptides were found to be conserved with minor variations. Prediction tools confirmed the ability of predicted peptides to bind with diverse HLA (HLA-A, HLA-B, HLA-DP, HLA-DQ and HLA-DR) alleles. CABS-dock results displayed that the average root mean square deviation (RMSD) value was less than three in majority of cases representing strong binding affinity with HLA alleles. Population coverage analysis predicted high coverage (> 85%) for expected immune response in four continents (Africa, America, Asia and Europe). Nine out of ten blood samples exhibited enhanced IFN-γ secretion for two peptides (P2 and P3). Thus, the identified NP peptides can be considered as potential synthetic vaccine candidates against Ebola virus.


Epitope-based vaccine Conservation analysis HLA alleles Molecular docking Ebola nucleoprotein 



We express our sincere thanks to the scientific community for developing in silico tools. We also express our gratitude towards Dr. Akshey Jain and Dr. Vandana Singla for providing us blood samples of healthy volunteers.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (PDF 17 KB)
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Supplementary material 2 (PDF 99 KB)
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Supplementary material 3 (PDF 20 KB)
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Supplementary material 4 (PDF 67 KB)


  1. 1.
    Kuhn JH, Becker S, Ebihara H et al (2010) Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations. Arch Virol 155(12):2083–2103Google Scholar
  2. 2.
    Lee JE, Fusco ML, Hessell AJ, Oswald WB, Burton DR, Saphire EO (2008) Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 454(7201):177–182Google Scholar
  3. 3.
    Sanchez A, Lukwiya M, Bausch D, Mahanty S, Sanchez AJ, Wagoner KD, Rollin PE (2004) Analysis of human peripheral blood samples from fatal and nonfatal cases of Ebola (Sudan) hemorrhagic fever: cellular responses, virus load, and nitric oxide levels. J Virol 78(19):10370–10377Google Scholar
  4. 4.
    Ilesanmi O, Alele FO (2016) Knowledge, attitude and perception of Ebola virus disease among secondary school students in Ondo State, Nigeria, October, 2014. PLoS Curr 8: ecurrents.outbreaks.c04b88cd5cd03cccb99e125657eecd76Google Scholar
  5. 5.
    Crisis update—November (2018) Accessed 5 Jan 2019
  6. 6.
    Brauburger K, Boehmann Y, Tsuda Y, Hoenen T, Olejnik J, Schumann M, Ebihara H, Muhlberger E (2014) Analysis of the highly diverse gene borders in Ebola virus reveals a distinct mechanism of transcriptional regulation. J Virol 88(21):12558–12571Google Scholar
  7. 7.
    Sun Y, Guo Y, Lou Z (2012) A versatile building block: the structures and functions of negative-sense single-stranded RNA virus nucleocapsid proteins. Protein Cell 3(12):893–902Google Scholar
  8. 8.
    Huang Y, Xu L, Sun Y, Nabel GJ (2002) The assembly of Ebola virus nucleocapsid requires virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein. Mol Cell 10(2):307–316Google Scholar
  9. 9.
    Trunschke M, Conrad D, Enterlein S, Olejnik J, Brauburger K, Muhlberger E (2013) The L-VP35 and L-L interaction domains reside in the amino terminus of the Ebola virus L protein and are potential targets for antivirals. Virology 441(2):135–145Google Scholar
  10. 10.
    Su Z, Wu C, Shi L et al (2018) Electron cryo-microscopy structure of Ebola virus nucleoprotein reveals a mechanism for nucleocapsid-like assembly. Cell 172(5):966–978Google Scholar
  11. 11.
    Kirchdoerfer RN, Abelson DM, Li S, Wood MR, Saphire EO (2015) Assembly of the Ebola virus nucleoprotein from a chaperoned VP35 complex. Cell Rep 12(1):140–149Google Scholar
  12. 12.
    Dong S, Yang P, Li G, Liu B, Wang W, Liu X, Xia B, Yang C, Lou Z, Guo Y, Rao Z (2015) Insight into the Ebola virus nucleocapsid assembly mechanism: crystal structure of Ebola virus nucleoprotein core domain at 1.8 Å resolution. Protein Cell 6(5):351–362Google Scholar
  13. 13.
    Wilson JA, Bray M, Bakken R, Hart MK (2001) Vaccine potential of Ebola virus VP24, VP30, VP35, and VP40 proteins. Virology 286(2):384–390Google Scholar
  14. 14.
    Sakabe S, Sullivan BM, Hartnett JN et al (2018) Analysis of CD8+ T cell response during the 2013–2016 Ebola epidemic in West Africa. Proc Natl Acad Sci USA 115(32):E7578–E7586Google Scholar
  15. 15.
    Herbert AS, Kuehne AI, Barth JF et al (2013) Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus. J Virol 87(9):4952–4964Google Scholar
  16. 16.
    Swenson DL, Wang D, Luo M, Warfield KL, Woraratanadharm J, Holman DH, Dong JY, Pratt WD (2008) Vaccine to confer to nonhuman primates complete protection against multistrain Ebola and Marburg virus infections. Clin Vccine Immunol 15(3):460–467Google Scholar
  17. 17.
    Martins KA, Jahrling PB, Bavari S, Kuhn JH (2016) Ebola virus disease candidate vaccines under evaluation in clinical trials. Expert Rev Vaccines 15(9):1101–1112Google Scholar
  18. 18.
    Lohia N, Baranwal M (2014) Conserved peptide containing overlapping CD4 + and CD8 + T cell epitopes in H1N1 influenza virus: An immunoinformatics approach. Viral Immunol 27(5):225–234Google Scholar
  19. 19.
    Sirskyj D, Diaz-Mitoma F, Golshani A, Kumar A, Azizi A (2011) Innovative bioinformatic approaches for developing peptide-based vaccines against hypervariable viruses. Immunol Cell Biol 89(1):81–89Google Scholar
  20. 20.
    Hossain MU, Keya CA, Das KC, Hashem A, Omar TM, Khan MA, Rakib-Uz-Zaman SM, Salimullah M (2018) An immunopharmacoinformatics approach in development of vaccine and drug candidates for West Nile Virus. Front Chem 6(246).
  21. 21.
    Ali MT, Islam MO (2015) A highly conserved GEQYQQLR epitope has been identified in the nucleoprotein of Ebola virus by using an in silico approach. Adv Bioinformatics 2015:278197. Google Scholar
  22. 22.
    Lohia N, Baranwal M (2017) Immune response of highly conserved influenza A virus matrix 1 peptides: matrix 1 peptides for influenza vaccine. Microbiol Immunol 61(6):225–231Google Scholar
  23. 23.
    Afley P, Dohre SK, Prasad GB, Kumar S (2015) Prediction of T cell epitopes of Brucella abortus and evaluation of their protective role in mice. Appl Microbiol Biotechnol 99(18):7625–7637Google Scholar
  24. 24.
    Bounds CE, Terry FE, Moise L, Hannaman D, Martin WD, De Groot AS, Suschak JJ, Dupuy LC, Schmaljohn CS (2017) An immunoinformatics-derived DNA vaccine encoding human class II T cell epitopes of Ebola virus, Sudan virus, and Venezuelan equine encephalitis virus is immunogenic in HLA transgenic mice. Hum Vaccin Immunother 13(12):2824–2836Google Scholar
  25. 25.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797Google Scholar
  26. 26.
    Miotto O, Heiny A, Tan TW, August JT, Brusic V (2008) Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by large scale mutual information analysis. BMC Bioinformatics 9(1):18Google Scholar
  27. 27.
    Dhiman G, Lohia N, Jain S, Baranwal M (2016) Metadherin peptides containing CD4+ and CD8+ T cell epitopes as a therapeutic vaccine candidate against cancer. Microbiol Immunol 60(9):646–652Google Scholar
  28. 28.
    Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50(3–4):213–219Google Scholar
  29. 29.
    Larsen MV, Lundegaard C, Lamberth K, Buus S, Lund O, Nielsen M (2007) Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction. BMC Bioinformatics 8:424Google Scholar
  30. 30.
    Nielsen M, Lundegaard C, Lund O (2007) Prediction of HLA class II binding affinity using SMMalign, a novel stabilization matrix alignment method. BMC Bioinformatics 8:238Google Scholar
  31. 31.
    Bhasin M, Raghava GP (2004) SVM based method for predicting HLA-DRB1*0401 binding peptides in an antigen sequence. Bioinformatics 20(3):421–423Google Scholar
  32. 32.
    Singh H, Raghava GP (2001) ProPred: prediction of HLA-DR binding sites. Bioinformatics 17(12):1236–1237Google Scholar
  33. 33.
    Saha S, Raghava GP (2006) Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins 65(1):40–48Google Scholar
  34. 34.
    Saha S, Raghava GPS (2006) AlgPred: prediction of allergenic proteins and mapping of IgE epitopes. Nucleic Acids Res 34:W202–W209Google Scholar
  35. 35.
    Kurcinski M, Jamroz M, Blaszczyk M, Kolinski A, Kmiecik S (2015) CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site. Nucleic Acids Res 43(W1):W419–W424Google Scholar
  36. 36.
    Lohia N, Baranwal M (2018) Highly conserved hemagglutinin peptides of H1N1 influenza virus elicit immune response. 3 Biotech 8(12):492. Google Scholar
  37. 37.
    Arango MT, Perricone C, Kivity S, Cipriano E, Ceccarelli F, Valesini G, Shoenfeld Y (2017) HLA-DRB1 the notorious gene in the mosaic of autoimmunity. Immunol Res 65(1):82–98Google Scholar
  38. 38.
    Nikbin B, Nicknam MH, Hadinedoushan H, Ansaripour B, Moradi B, Yekaninejad M, Aminikhah M, Ranjbar MM, Amirzargar A (2017) Human leukocyte antigen (HLA) class I and II polymorphism in Iranian healthy population from Yazd Province. Iran J Allergy Asthma Immunol 16(1):1–13Google Scholar
  39. 39.
    Wieczorek M, Abualrous ET, Sticht J, Álvaro-Benito M, Stolzenberg S, Noé F, Freund C (2017) Major histocompatibility complex (MHC) class I and MHC class II proteins: conformational plasticity in antigen presentation. Front Immunol 8(292).
  40. 40.
    Blaszczyk M, Kurcinski M, Kouza M, Wieteska L, Debinski A, Kolinski A, Kmiecik S (2016) Modeling of protein-peptide interactions using the CABS-dock web server for binding site search and flexible docking. Methods 93:72–83Google Scholar
  41. 41.
    Li W, Joshi MD, Singhania S, Ramsey KH, Murtl AK (2014) Peptide vaccine: progress and challenges. Vaccines 2:515–536Google Scholar
  42. 42.
    Francis JN, Bunce CJ, Horlock C, Watson JM, Warrington SJ, Georges B, Brown CB (2015) A novel peptide-based pan-influenza A vaccine: a double blind, randomised clinical trial of immunogenicity and safety. Vaccine 33(2):396–402Google Scholar
  43. 43.
    Zom GG, Welters MJP, Loof NM et al (2016) TLR2 ligand-synthetic long peptide conjugates effectively stimulate tumor-draining lymph node T cells of cervical cancer patients. Oncotarget 7(41):67087–67100Google Scholar
  44. 44.
    Agallou M, Athanasiou E, Koutsoni O, Dotsika E, Karagouni E (2014) Experimental validation of multi-epitope peptides including promising MHC Class I- and II-restricted epitopes of four known Leishmania infantum proteins. Front Immunol 5(268).
  45. 45.
    Vani J, Shaila MS, Chandra NR, Nayak R (2006) A combined immuno-informatics and structure-based modeling approach for prediction of T cell epitopes of secretory proteins of Mycobacterium tuberculosis. Microbes Infect 8(3):738–746Google Scholar
  46. 46.
    Lohia N, Baranwal M (2015) Identification of conserved peptides comprising multiple T cell epitopes of matrix 1 protein in H1N1 influenza virus. Viral Immunol 28(10):570–579Google Scholar
  47. 47.
    Dikhit MR, Kumar A, Das S, Dehury B, Rout AK, Jamal F, Sahoo GC, Topno RK, Pandey K, Das VNR, Bimal S, Das P (2017) Identification of Potential MHC Class-II-restricted epitopes derived from Leishmania donovani antigens by reverse vaccinology and evaluation of Their CD4 + T-cell responsiveness against visceral leishmaniasis. Front Immunol 8(1763).
  48. 48.
    Yassin GM, Amin MA, Attia AS (2016) Immunoinformatics identifies a lactoferrin binding protein a peptide as a promising vaccine with a global protective prospective against moraxella catarrhalis. J Infect Dis 213(12):1938–1945Google Scholar
  49. 49.
    Sundar K, Boesen A, Coico R (2007) Computational prediction and identification of HLA-A2.1-specific Ebola virus CTL epitopes. Virology 360(2):257–263Google Scholar
  50. 50.
    Dutta DK, Rhodes K, Wood SC (2015) In silico prediction of Ebola Zaire GP(1,2) immuno-dominant epitopes for the Balb/c mouse. BMC Immunol 16:59. Google Scholar
  51. 51.
    Dikhit MR, Kumar S, Vijaymahantesh et al (2015) Computational elucidation of potential antigenic CTL epitopes in Ebola virus. Infection Genetics and Evolution 36:369–375Google Scholar
  52. 52.
    Ruibal P, Oestereich L, Lüdtke A et al (2016) Unique human immune signature of Ebola virus disease in Guinea. Nature 533(7601):100–104Google Scholar
  53. 53.
    Theaker SM, Rius C, Greenshields-Watson A, Lloyd A, Trimby A, Fuller A, Miles JJ, Cole DK, Peakman M, Sewell AK, Dolton G (2016) T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones. J Immunol Methods 430:43–50Google Scholar
  54. 54.
    Gupta M, Greer P, Mahanty S, Shieh WJ, Zaki SR, Ahmed R, Rollin PE (2005) CD8-Mediated Protection against Ebola Virus Infection Is Perforin Dependent. J Immunol 174(7):4198–4202Google Scholar
  55. 55.
    Simmons G, Lee A, Rennekamp AJ, Fan X, Bates P, Shen H (2004) Identification of murine T-cell epitopes in Ebola virus nucleoprotein. Virology 318(1):224–230Google Scholar
  56. 56.
    Wilson JA, Hart MK (2001) Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein. J Virol 75(6):2660–2664Google Scholar
  57. 57.
    Fritsche PJ, Helbling A, Ballmer-Weber BK (2010) Vaccine hypersensitivity—update and overview. Swiss Med Wkly 140(17–18):238–246Google Scholar
  58. 58.
    Cohen AD, Shoenfeld Y (1996) Vaccine-induced autoimmunity. J Autoimmun 9(6):699–703Google Scholar
  59. 59.
    Meyboom RH, Fucik H, Edwards IR (1995) Thrombocytopenia reported in association with hepatitis B and A vaccines. Lancet 345(8965):1638Google Scholar
  60. 60.
    Topaloglu H, Berker M, Kansu T, Saatci U, Renda Y (1992) Optic neuritis and myelitis after booster tetanus toxoid vaccination. Lancet 339(8786):178–179Google Scholar
  61. 61.
    Patja A, Mäkinen-Kiljunen S, Davidkin I, Paunio M, Peltola H (2001) Allergic reactions to measles-mumps-rubella vaccination. Pediatrics 107(2):E27Google Scholar
  62. 62.
    Businco L (1994) Measles, mumps, rubella immunization in egg-allergic children: a long-lasting debate. Ann Allergy 72(1):1–3Google Scholar
  63. 63.
    Atsmon J, Kate-Ilovitz E, Shaikevich D, Singer Y, Volokhov I, Haim KY, Ben-Yedidia T (2012) Safety and immunogenicity of multimeric-001–a novel universal influenza vaccine. J Clin Immunol 32(3):595–603Google Scholar
  64. 64.
    Hou Y, Guo Y, Wu C, Shen N, Jiang Y, Wang J (2012) Prediction and identification of T cell epitopes in the H5N1 influenza virus nucleoprotein in chicken. PLoS One 7(6):e39344Google Scholar
  65. 65.
    Tawar RG, Duquerroy S, Vonrhein C et al (2009) Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 326(5957):1279–1283Google Scholar
  66. 66.
    Yabukarski F, Lawrence P, Tarbouriech N, Bourhis JM, Delaforge E, Jensen MR, Ruigrok RW, Blackledge M, Volchkov V, Jamin M (2014) Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone. Nat Struct Mol Biol 21(9):754–759Google Scholar
  67. 67.
    Alayyoubi M, Leser GP, Kors CA, Lamb RA (2015) Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein-RNA complex. Proc Natl Acad Sci USA 112(14):E1792–E1799Google Scholar
  68. 68.
    He Y, Li J, Mao W et al (2018) HLA common and well-documented (CWD) alleles in China. HLA Google Scholar
  69. 69.
    Robinson J, Halliwell JA, Hayhurst JD, Flicek P, Parham P, Marsh SG (2015) The IPD and IMGT/HLA database: allele variant databases. Nucleic Acids Res 43:423–431Google Scholar
  70. 70.
    Kalyanaraman N (2018) In silico prediction of potential vaccine candidates on capsid protein of human bocavirus 1. Mol Immunol 93:193–205Google Scholar
  71. 71.
    Watanabe S, Noda T, Kawaoka Y (2006) Functional mapping of the nucleoprotein of Ebola virus. J Virol 80(8):3743–3751Google Scholar
  72. 72.
    Albertini AA, Wernimont AK, Muziol T, Ravelli RB, Clapier CR, Schoehn G, Weissenhorn W, Ruigrok RW (2006) Crystal structure of the rabies virus nucleoprotein-RNA complex. Science 313(5785):360–363Google Scholar
  73. 73.
    Noda T, Hagiwara K, Sagara H, Kawaoka Y (2010) Characterization of the Ebola virus nucleoprotein-RNA complex. J Gen Virol 91(6):1478–1483Google Scholar
  74. 74.
    Medaglini D (2018) Correlates of vaccine-induced protective immunity against Ebola virus disease. Semin Immunol. Google Scholar
  75. 75.
    Henao-Restrepo AM, Camacho A, Longini IM et al (2017) Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!). Lancet 389(10068):505–518Google Scholar
  76. 76.
    Halperin SA, Arribas JR, Rupp R, Andrews CP, Chu L, Das R, Simon JK, Onorato MT, Liu K, Martin J, Helmond FA (2017) Six-month safety data of recombinant vesicular stomatitis virus-zaire ebola virus envelope glycoprotein vaccine in a phase 3 double-blind, placebo-controlled randomized study in healthy adults. J infect Dis 215(12):1789–1798Google Scholar
  77. 77.
    Ledgerwood JE, DeZure AD, Stanley DA et al (2017) Chimpanzee Adenovirus Vector Ebola Vaccine. N Engl J Med 376(10):928–938Google Scholar
  78. 78.
    Ewer K, Rampling T, Venkatraman N et al (2016) A monovalent chimpanzee adenovirus ebola vaccine boosted with MVA. N Engl J Med 374(17):1635–1646Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyThapar Institute of Engineering and TechnologyPatialaIndia

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