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

Abstract

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(1,2) Fc 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(1,2) Fc.

Methods: The GP(1,2) Ebola 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(1,2) have 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.

Fig. 1

Stabilization of K^D expression IN K^D RMAS cells by Ebola GP_1,2 peptides. GP_1,2 Ebola Zaire, NCBI accession number AAB81004.1, was analyzed by both BIMAS and SYFPEITHI. The GP_1,2 protein sequence was divided into segments of nine amino acid that overlapped by eight amino acids. Peptides that scored better than >150 in BIMAS and >24 in SYFPEITHI were selected for further study. BD FACS SCAN was used to assess peptide stabilization of MHC expressed on K^D RMAS cells. The heavy line represents isotype control. The results are representative of three independent experiments

Fig. 2

Sequence alignment of GP_1,2 from Ebola Zaire (AAA96744.1), Sudan virus (AAP88031.1), Bundibugyo (YP_003815435), Tai Forest (YP_003815426.1), Reston (Q66799.1), and Marburg Lake Victoria (P35254.1). Clustal W alignments were performed and highlights were added via Boxshade. Amino acids that are identical are shaded black while chemically similar amino acids are grey. The location of peptides P8, P9 and P10 are indicated by the boxes. P8 is in a highly variable region while P9 and P10 are located in highly conserved regions for cytosolic entry and cellular recognition, respectively

Fig. 3

IFN-γ Elispot responses by splenocytes were evaluated following immunization with Ebola Zaire GP_1,2 peptides. Mice were immunized and boosted 7 days after initial immunization. Splenocytes were re-stimulated in vitro with peptide overnight. The mean IFN- γ Elispots/10⁶ splenocytes +/− the S.E. is presented. The results are representative of three independent experiments. Asterisks indicate statistically significant IFN- γ increase in peptide-exposed cells at 100 mm concentration compared with those cells treated with 0.1 mm peptide (* = p <0.05)

Fig. 4

Induction of intracellular IFN- γ production following immunization with P8, P9 and P10. Splenocytes from peptide immunized animals were cultured for 24 h in vitro with respective peptide. Cells were collected, washed, stained with CD3ε PerCP and anti-CD 8 FITC and fixed. The cells were further stained with anti-IFN-γ antibody and were analyzed using BD FACS Canto. The results are represented as mean +/− S.E. of three independent experiments. Asterisks indicate statistically significant IFN-γ increase in peptide-exposed cells compared with those untreated cells (* = p <0.05)

Fig. 5

Tetramer staining of immunized splencocytes. Splenocytes from peptide immunized animals were stained with K^D tetramers prepared with P8, P9, P10 or Adjuvant (mock-infected). The cells were counter stained with CD8 FITC and CD3 PerCP, then fixed. The stained cells were analyzed in a FACS ARIA. Tetramer positive cells were sorted and re-analyzed. Pre-sort and Post sorted tetramer positive cells are shown in figure. The results are representative of three independent experiments

Fig. 6

CTL response induced by peptides P8, P9, and P10. Splenocytes from immunized animals were cultured for 5 days in vitro with peptide, collected and counted. In parallel, K^D cells were incubated overnight with P8, P9, P10 or adjuvant (mock control), washed and then labeled with DDAO cell tracker. Effector and target cells were mixed and incubated for 4 h in the indicated ratios and fixed. The cells were stained with anti-cleaved Caspase-3 PE and analyzed by flow cytometry using a FACS Canto. The mean percentage of Caspase positive K^D cells +/− the SE is presented. The results are representative of three independent experiments. Asterisks indicate statistically significant increase in Caspase -3 positive cells compared with those Caspase positive cells in mock (* = p <0.05)

Fig. 7

CTL response induced by GP_1,2 immunization. Splenocytes from immunized animals were cultured for 5 days in vitro with P8, P9 and P10 peptides, for the expansion of peptide specific CTLs, that were collected and counted. In parallel, K^D cells were incubated overnight with P8, P9, P10 or adjuvant (mock control), washed and then labeled with DDAO cell tracker. Effector and target cells were mixed and incubated for 4 h in the indicated ratios and fixed. The cells were then stained with anti-cleaved Caspase-3 PE and analyzed by flow cytometers, using a FACS Canto. The mean percentage of Caspase positive K^D cells +/− the SE is presented. The results are representative of three independent experiments. Asterisks indicate statistically significant increase in Caspase -3 positive cells compared with those Caspase-3 positive cells in mock (* = p < 0.05)

Fig. 8

Induction of intracellular IFN-γ production following immunization with GP_1,2-Fc. Splenocytes from GP_1,2-Fc immunized animals were cultured for 24 h in vitro with P8, P9 and P10 peptides. Cells were collected, washed, stained with CD3ε PerCP and anti-CD 8 FITC and fixed. The cells were further stained with anti-IFN-γ antibody and were analyzed using BD FACS Canto. The results are represented as mean +/− S.E. of two independent experiments