In silico prediction of Ebola Zaire GP 1,2 immuno-dominant epitopes for the Balb/c mouse

BMC Immunology

Steven C. Wood²  Kelly Rhodes¹  Debargh K. Dutta³ 

[1]University of Maryland, College Park 20742, MD, USA

[2]Division of Biology, Chemistry and Materials Science, Center for Devices and Radiological Health, FDA, Silver Spring 20993, MD, USA

[3]Department of Medicine, USUHS, 4301 Jones Bridge road, Bethesda 20814, MD, USA

关键词: Tetramer;    CTL;    Immunodominant epitopes;    GP1,2;    Ebola;   

Others  :  1228733
DOI  :  10.1186/s12865-015-0126-8

 received in 2015-06-10, accepted in 2015-10-01,  发布年份 2015

【 摘 要 】

Background

Ebola is a Filovirus (FV) that induces a highly communicable and deadly hemorrhagic fever. Currently, there are no approved vaccines to treat FV infections. Protection from FV infection requires cell mediated and humoral immunity. Glycoprotein GP _1,2Fc Zaire, a recombinant FV human Fc fusion protein, has been shown to confer protection against mouse adapted Zaire Ebola virus. The present studies are focused upon identifying immunodominant epitopes using in silico methods and developing tetramers as a diagnostic reagent to detect cell mediated immune responses to GP _1,2Fc.

Methods

The GP _1,2Ebola Zaire sequence from the 1976 outbreak was analyzed by both BIMAS and SYFPEITHI algorithms to identify potential immuno-dominant epitopes. Several peptides were synthesized and screened in flow-based MHC stability studies. Three candidate peptides, P8, P9 and P10, were identified and, following immunization in Balb/c mice, all three peptides induced IFN-γ as detected by ELISpot and intracellular staining.

Results

Significantly, P8, P9 and P10 generated robust cytotoxic T-cell responses (CTL) as determined by a flow cytometry-based Caspase assay. Antigen specific cells were also detected, using tetramers. Both P9 and P10 have sequence homology with highly conserved regions of several strains of FV.

Conclusions

In sum, three immunodominant sequences of the Ebola GP _1,2have been identified using in silico methods that may confer protection against mouse adapted Ebola Zaire. The development of tetramer reagents will provide unique insight into the potency and durability of medical countermeasure vaccines for known bioterrorism threat agents in preclinical models.

【 授权许可】

   
2015 Dutta et al.

【 图 表 】

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【 参考文献 】

• [1]Kortepeter MG, Bausch DG, Bray M. Basic clinical and laboratory features of filoviral hemorrhagic fever. J Infect Dis. 2011; 204 Suppl 3:S810-816.
• [2]Warfield KL, Bosio CM, Welcher BC, Deal EM, Mohamadzadeh M, Schmaljohn A, Aman MJ, Bavari S. Ebola virus-like particles protect from lethal Ebola virus infection. Proc Natl Acad Sci U S A. 2003; 100(26):15889-15894.
• [3]Baize S, Leroy EM, Georges-Courbot MC, Capron M, Lansoud-Soukate J, Debre P, Fisher-Hoch SP, McCormick JB, Georges AJ. Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. Nat Med. 1999; 5(4):423-426.
• [4]Bradfute SB, Warfield KL, Bavari S. Functional CD8+ T cell responses in lethal Ebola virus infection. J Immunol. 2008; 180(6):4058-4066.
• [5]Oswald WB, Geisbert TW, Davis KJ, Geisbert JB, Sullivan NJ, Jahrling PB, Parren PW, Burton DR. Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys. PLoS Pathog. 2007; 3(1):e9.
• [6]Lee JE, Fusco ML, Hessell AJ, Oswald WB, Burton DR, Saphire EO. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature. 2008; 454(7201):177-182.
• [7]Warfield KL, Olinger GG. Protective role of cytotoxic T lymphocytes in filovirus hemorrhagic fever. J Biomed Biotechnol. 2011; 2011:984241.
• [8]Gupta M, Greer P, Mahanty S, Shieh WJ, Zaki SR, Ahmed R, Rollin PE. CD8-mediated protection against Ebola virus infection is perforin dependent. J Immunol. 2005; 174(7):4198-4202.
• [9]Rao M, Matyas GR, Grieder F, Anderson K, Jahrling PB, Alving CR. Cytotoxic T lymphocytes to Ebola Zaire virus are induced in mice by immunization with liposomes containing lipid A. Vaccine. 1999; 17(23-24):2991-2998.
• [10]Olinger GG, Bailey MA, Dye JM, Bakken R, Kuehne A, Kondig J, Wilson J, Hogan RJ, Hart MK. Protective cytotoxic T-cell responses induced by venezuelan equine encephalitis virus replicons expressing Ebola virus proteins. J Virol. 2005; 79(22):14189-14196.
• [11]Sullivan NJ, Sanchez A, Rollin PE, Yang ZY, Nabel GJ. Development of a preventive vaccine for Ebola virus infection in primates. Nature. 2000; 408(6812):605-609.
• [12]Mire CE, Geisbert JB, Marzi A, Agans KN, Feldmann H, Geisbert TW. Vesicular stomatitis virus-based vaccines protect nonhuman primates against Bundibugyo ebolavirus. PLoS Negl Trop Dis. 2013; 7(12):e2600.
• [13]DiNapoli JM, Yang LJ, Samal SK, Murphy BR, Collins PL, Bukreyev A. Respiratory tract immunization of non-human primates with a Newcastle disease virus-vectored vaccine candidate against Ebola virus elicits a neutralizing antibody response. Vaccine. 2010; 29(1):17-25.
• [14]Papaneri AB, Wirblich C, Cann JA, Cooper K, Jahrling PB, Schnell MJ, Blaney JE. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence. Virology. 2012; 434(1):18-26.
• [15]Konduru K, Bradfute SB, Jacques J, Manangeeswaran M, Nakamura S, Morshed S, Wood SC, Bavari S, Kaplan GG. Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice. Vaccine. 2011; 29(16):2968-2977.
• [16]Dowling W, Thompson E, Badger C, Mellquist JL, Garrison AR, Smith JM, Paragas J, Hogan RJ, Schmaljohn C. Influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus GP DNA vaccines. J Virol. 2007; 81(4):1821-1837.
• [17]Warfield KL, Olinger G, Deal EM, Swenson DL, Bailey M, Negley DL, Hart MK, Bavari S. Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection. J Immunol. 2005; 175(2):1184-1191.
• [18]Wu S, Yu T, Song X, Yi S, Hou L, Chen W. Prediction and identification of mouse cytotoxic T lymphocyte epitopes in Ebola virus glycoproteins. Virol J. 2012; 9:111. BioMed Central Full Text
• [19]Rao M, Bray M, Alving CR, Jahrling P, Matyas GR. Induction of immune responses in mice and monkeys to Ebola virus after immunization with liposome-encapsulated irradiated Ebola virus: protection in mice requires CD4(+) T cells. J Virol. 2002; 76(18):9176-9185.
• [20]Boesen A, Sundar K, Coico R. Lassa fever virus peptides predicted by computational analysis induce epitope-specific cytotoxic-T-lymphocyte responses in HLA-A2.1 transgenic mice. Clin Diagn Lab Immunol. 2005; 12(10):1223-1230.
• [21]Sundar K, Boesen A, Coico R. Computational prediction and identification of HLA-A2.1-specific Ebola virus CTL epitopes. Virology. 2007; 360(2):257-263.
• [22]Walker LJ, Sewell AK, Klenerman P. T cell sensitivity and the outcome of viral infection. Clin Exp Immunol. 2010; 159(3):245-255.
• [23]Sims S, Willberg C, Klenerman P. MHC-peptide tetramers for the analysis of antigen-specific T cells. Expert Rev Vaccines. 2010; 9(7):765-774.
• [24]Wong G, Richardson JS, Pillet S, Patel A, Qiu X, Alimonti J, Hogan J, Zhang Y, Takada A, Feldmann H et al.. Immune parameters correlate with protection against ebola virus infection in rodents and nonhuman primates. Sci Transl Med. 2012; 4(158):158ra146.
• [25]Marzi A, Engelmann F, Feldmann F, Haberthur K, Shupert WL, Brining D, Scott DP, Geisbert TW, Kawaoka Y, Katze MG et al.. Antibodies are necessary for rVSV/ZEBOV-GP-mediated protection against lethal Ebola virus challenge in nonhuman primates. Proc Natl Acad Sci U S A. 2013; 110(5):1893-1898.
• [26]Dye JM, Herbert AS, Kuehne AI, Barth JF, Muhammad MA, Zak SE, Ortiz RA, Prugar LI, Pratt WD. Postexposure antibody prophylaxis protects nonhuman primates from filovirus disease. Proc Natl Acad Sci U S A. 2012; 109(13):5034-5039.
• [27]Klinman DM, Nutman TB. ELISPOT assay to detect cytokine-secreting murine and human cells. Curr Protoc Immunol. 2001; Chapter 6:Unit 6 19.
• [28]Klinman D. ELISPOT assay to detect cytokine-secreting murine and human cells. Curr Protoc Immunol. 2008; Chapter 6:Unit 6 19.
• [29]Salerno-Goncalves R, Pasetti MF, Sztein MB. Characterization of CD8(+) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine. J Immunol. 2002; 169(4):2196-2203.
• [30]Gillespie G, Mutis T, Schrama E, Kamp J, Esendam B, Falkenburg JF, Goulmy E, Moss P. HLA class I-minor histocompatibility antigen tetramers select cytotoxic T cells with high avidity to the natural ligand. Hematol J. 2000; 1(6):403-410.
• [31]Kostense S, Ogg GS, Manting EH, Gillespie G, Joling J, Vandenberghe K, Veenhof EZ, van Baarle D, Jurriaans S, Klein MR et al.. High viral burden in the presence of major HIV-specific CD8(+) T cell expansions: evidence for impaired CTL effector function. Eur J Immunol. 2001; 31(3):677-686.
• [32]Lechner F, Wong DK, Dunbar PR, Chapman R, Chung RT, Dohrenwend P, Robbins G, Phillips R, Klenerman P, Walker BD. Analysis of successful immune responses in persons infected with hepatitis C virus. J Exp Med. 2000; 191(9):1499-1512.
• [33]Murali-Krishna K, Altman JD, Suresh M, Sourdive D, Zajac A, Ahmed R. In vivo dynamics of anti-viral CD8 T cell responses to different epitopes. An evaluation of bystander activation in primary and secondary responses to viral infection. Adv Exp Med Biol. 1998; 452:123-142.
• [34]He LW, Hakimi J, Salha D, Miron I, Dunn P, Radvanyi L. A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells. J Immunol Methods. 2005; 304(1-2):43-59.
• [35]Geisbert TW, Pushko P, Anderson K, Smith J, Davis KJ, Jahrling PB. Evaluation in nonhuman primates of vaccines against Ebola virus. Emerg Infect Dis. 2002; 8(5):503-507.