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Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells.

Ternette N, Block PD, Sánchez-Bernabéu Á, Borthwick N, Pappalardo E, Abdul-Jawad S, Ondondo B, Charles PD, Dorrell L, Kessler BM, Hanke T - J. Virol. (2015)

Bottom Line: We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with the class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 were derived from the HIVconsv immunogen.MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance.Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.

View Article: PubMed Central - PubMed

Affiliation: The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom nicola.ternette@ndm.ox.ac.uk.

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Responses in humans vaccinated with HIVconsv recognizing eluted, MS-identified peptides. (A) Schematic view of the HIVconsv immunogen, with the 14 conserved regions of HIV-1 that were combined in the immunogen represented as colored boxes and with the original HIV-1 region stated (Gag, Pol, Vif, and Env). Letters above the boxes (A, B, C, and D) indicate the clade of origin. The positions of eluted peptides identified in the HIVconsv immunogen are indicated by red bars. Genomic regions of the HXB2 strain are shown as gray rectangles. TAGs, epitope tag sequences; LTR, long terminal repeat; nt, nucleotides. (B) The magnitude of the response (in SFU/106 PMBCs) is plotted for each individual, summed for all 15-mer peptides containing MS-identified sequences (left y axis), in addition to the percent magnitude compared to the total magnitude of responses to all 15-mer peptides spanning the full HIVconsv immunogen (right y axis). The breadth of the response is indicated by the number of peptides generating a response in each individual subject (MS-identified/total number of peptides, indicated above each bar). Individual responses to the indicated peptide sequences are shown below each graph column, and HLA genotypes for each volunteer are indicated. Matching HLA types are highlighted in green, and matching alleles from an identical HLA supertype are highlighted in violet. P, placebo recipient.
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Figure 5: Responses in humans vaccinated with HIVconsv recognizing eluted, MS-identified peptides. (A) Schematic view of the HIVconsv immunogen, with the 14 conserved regions of HIV-1 that were combined in the immunogen represented as colored boxes and with the original HIV-1 region stated (Gag, Pol, Vif, and Env). Letters above the boxes (A, B, C, and D) indicate the clade of origin. The positions of eluted peptides identified in the HIVconsv immunogen are indicated by red bars. Genomic regions of the HXB2 strain are shown as gray rectangles. TAGs, epitope tag sequences; LTR, long terminal repeat; nt, nucleotides. (B) The magnitude of the response (in SFU/106 PMBCs) is plotted for each individual, summed for all 15-mer peptides containing MS-identified sequences (left y axis), in addition to the percent magnitude compared to the total magnitude of responses to all 15-mer peptides spanning the full HIVconsv immunogen (right y axis). The breadth of the response is indicated by the number of peptides generating a response in each individual subject (MS-identified/total number of peptides, indicated above each bar). Individual responses to the indicated peptide sequences are shown below each graph column, and HLA genotypes for each volunteer are indicated. Matching HLA types are highlighted in green, and matching alleles from an identical HLA supertype are highlighted in violet. P, placebo recipient.

Mentions: To increase the discovery of HIVconsv-derived epitopes, HLA class I-associated peptides were eluted from Jurkat cells 6 h after MVA.HIVconsv infection in two separate experiments. This longer infection led to the identification of four additional sequences, totaling 11 HIVconsv-derived peptides (Table 1 and Fig. 4 and 5A). In order to validate the identified HIVconsv-derived peptide sequences, the corresponding synthetic peptide standards were analyzed under identical conditions, and the obtained fragment spectra were compared. Spectral comparisons for 6 out of the 11 peptides identified are depicted in Fig. 4.


Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells.

Ternette N, Block PD, Sánchez-Bernabéu Á, Borthwick N, Pappalardo E, Abdul-Jawad S, Ondondo B, Charles PD, Dorrell L, Kessler BM, Hanke T - J. Virol. (2015)

Responses in humans vaccinated with HIVconsv recognizing eluted, MS-identified peptides. (A) Schematic view of the HIVconsv immunogen, with the 14 conserved regions of HIV-1 that were combined in the immunogen represented as colored boxes and with the original HIV-1 region stated (Gag, Pol, Vif, and Env). Letters above the boxes (A, B, C, and D) indicate the clade of origin. The positions of eluted peptides identified in the HIVconsv immunogen are indicated by red bars. Genomic regions of the HXB2 strain are shown as gray rectangles. TAGs, epitope tag sequences; LTR, long terminal repeat; nt, nucleotides. (B) The magnitude of the response (in SFU/106 PMBCs) is plotted for each individual, summed for all 15-mer peptides containing MS-identified sequences (left y axis), in addition to the percent magnitude compared to the total magnitude of responses to all 15-mer peptides spanning the full HIVconsv immunogen (right y axis). The breadth of the response is indicated by the number of peptides generating a response in each individual subject (MS-identified/total number of peptides, indicated above each bar). Individual responses to the indicated peptide sequences are shown below each graph column, and HLA genotypes for each volunteer are indicated. Matching HLA types are highlighted in green, and matching alleles from an identical HLA supertype are highlighted in violet. P, placebo recipient.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4442425&req=5

Figure 5: Responses in humans vaccinated with HIVconsv recognizing eluted, MS-identified peptides. (A) Schematic view of the HIVconsv immunogen, with the 14 conserved regions of HIV-1 that were combined in the immunogen represented as colored boxes and with the original HIV-1 region stated (Gag, Pol, Vif, and Env). Letters above the boxes (A, B, C, and D) indicate the clade of origin. The positions of eluted peptides identified in the HIVconsv immunogen are indicated by red bars. Genomic regions of the HXB2 strain are shown as gray rectangles. TAGs, epitope tag sequences; LTR, long terminal repeat; nt, nucleotides. (B) The magnitude of the response (in SFU/106 PMBCs) is plotted for each individual, summed for all 15-mer peptides containing MS-identified sequences (left y axis), in addition to the percent magnitude compared to the total magnitude of responses to all 15-mer peptides spanning the full HIVconsv immunogen (right y axis). The breadth of the response is indicated by the number of peptides generating a response in each individual subject (MS-identified/total number of peptides, indicated above each bar). Individual responses to the indicated peptide sequences are shown below each graph column, and HLA genotypes for each volunteer are indicated. Matching HLA types are highlighted in green, and matching alleles from an identical HLA supertype are highlighted in violet. P, placebo recipient.
Mentions: To increase the discovery of HIVconsv-derived epitopes, HLA class I-associated peptides were eluted from Jurkat cells 6 h after MVA.HIVconsv infection in two separate experiments. This longer infection led to the identification of four additional sequences, totaling 11 HIVconsv-derived peptides (Table 1 and Fig. 4 and 5A). In order to validate the identified HIVconsv-derived peptide sequences, the corresponding synthetic peptide standards were analyzed under identical conditions, and the obtained fragment spectra were compared. Spectral comparisons for 6 out of the 11 peptides identified are depicted in Fig. 4.

Bottom Line: We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with the class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 were derived from the HIVconsv immunogen.MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance.Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.

View Article: PubMed Central - PubMed

Affiliation: The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom nicola.ternette@ndm.ox.ac.uk.

Show MeSH
Related in: MedlinePlus