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HIV-1 envelope resistance to proteasomal cleavage: implications for vaccine induced immune responses.

Steers NJ, Ratto-Kim S, de Souza MS, Currier JR, Kim JH, Michael NL, Alving CR, Rao M - PLoS ONE (2012)

Bottom Line: The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively.Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters.The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4(+) T-cell lines-derived from RV144 vaccinees.

View Article: PubMed Central - PubMed

Affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America.

ABSTRACT

Background: Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response.

Methods: In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4(+) T-cell lines derived from RV144 volunteers.

Results: Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4(+) T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial.

Conclusions: Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4(+)T cell and antibody responses in the RV144 vaccinees.

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Related in: MedlinePlus

CAT cleavage maps of Env-A244.Env-A244 was subjected to CAT digestion for 90 min and 16 hrs. The peptide fragments were separated by UFLC, analyzed on an LCMS-IT-TOF mass spectrometer, and then identified using the MASCOT database. The numbering of Env-A244 is based on HXB2 numbering. Env-A244 also contains a gD tag at its N-terminus, the sequence of the gD tag is not shown. The different regions of Env-A244 (C1, V1, V2, C2, V3, C3, V4, C4, V5, and C5) are denoted under the sequence. The Env-A244 peptides generated from CAT B, (blue line) CAT D (red line), CAT K (green line), CAT L (purple line), and CAT S (orange) are shown The solid and the dotted lines above the sequence represent the peptide fragments identified at the 90 min and the 16 hrs time point, respectively. These fragments could contain potential MHC class I and class II epitopes. The solid black lines below the sequence represent MHC class I epitopes, while the dashed black lines represent MHC class II epitopes as reported in the Las Alamos database.
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pone-0042579-g003: CAT cleavage maps of Env-A244.Env-A244 was subjected to CAT digestion for 90 min and 16 hrs. The peptide fragments were separated by UFLC, analyzed on an LCMS-IT-TOF mass spectrometer, and then identified using the MASCOT database. The numbering of Env-A244 is based on HXB2 numbering. Env-A244 also contains a gD tag at its N-terminus, the sequence of the gD tag is not shown. The different regions of Env-A244 (C1, V1, V2, C2, V3, C3, V4, C4, V5, and C5) are denoted under the sequence. The Env-A244 peptides generated from CAT B, (blue line) CAT D (red line), CAT K (green line), CAT L (purple line), and CAT S (orange) are shown The solid and the dotted lines above the sequence represent the peptide fragments identified at the 90 min and the 16 hrs time point, respectively. These fragments could contain potential MHC class I and class II epitopes. The solid black lines below the sequence represent MHC class I epitopes, while the dashed black lines represent MHC class II epitopes as reported in the Las Alamos database.

Mentions: The peptides derived from CAT B, D, K, L, and S degradation of Env-A244 at 90 min (solid lines above the Env-A244 sequence) and additional peptides generated at 16 hrs (dotted lines above the Env-A244 sequence) were identified by mass spectrometry (Figure 3). The MHC class I and class II epitopes for Env-A244 as reported in the Los Alamos database are shown respectively beneath the amino acid sequence as a solid or hatched black line. Each of the cathepsin Env-A244 degradations generated clusters of potential peptide MHC class I and class II precursor epitopes (Figure 3 and Table 1) with dense clusters found in the C1, C2, V3, C3, C4, and C5 regions of Env-A244 that contained the CD4, chemokine, and majority of the neutralizing antibody binding sites. Each of the CAT generated varying number of peptides. The number of peptides produced was relatively consistent at 90 min and at 16 hrs with the exception of CAT B and D (Table 1). The concentration of individual peptides derived from each of the CAT degradations of Env-A244 was not assessed. Therefore, there could be varying concentrations of antigenic peptides in each of the CAT degradations.


HIV-1 envelope resistance to proteasomal cleavage: implications for vaccine induced immune responses.

Steers NJ, Ratto-Kim S, de Souza MS, Currier JR, Kim JH, Michael NL, Alving CR, Rao M - PLoS ONE (2012)

CAT cleavage maps of Env-A244.Env-A244 was subjected to CAT digestion for 90 min and 16 hrs. The peptide fragments were separated by UFLC, analyzed on an LCMS-IT-TOF mass spectrometer, and then identified using the MASCOT database. The numbering of Env-A244 is based on HXB2 numbering. Env-A244 also contains a gD tag at its N-terminus, the sequence of the gD tag is not shown. The different regions of Env-A244 (C1, V1, V2, C2, V3, C3, V4, C4, V5, and C5) are denoted under the sequence. The Env-A244 peptides generated from CAT B, (blue line) CAT D (red line), CAT K (green line), CAT L (purple line), and CAT S (orange) are shown The solid and the dotted lines above the sequence represent the peptide fragments identified at the 90 min and the 16 hrs time point, respectively. These fragments could contain potential MHC class I and class II epitopes. The solid black lines below the sequence represent MHC class I epitopes, while the dashed black lines represent MHC class II epitopes as reported in the Las Alamos database.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412807&req=5

pone-0042579-g003: CAT cleavage maps of Env-A244.Env-A244 was subjected to CAT digestion for 90 min and 16 hrs. The peptide fragments were separated by UFLC, analyzed on an LCMS-IT-TOF mass spectrometer, and then identified using the MASCOT database. The numbering of Env-A244 is based on HXB2 numbering. Env-A244 also contains a gD tag at its N-terminus, the sequence of the gD tag is not shown. The different regions of Env-A244 (C1, V1, V2, C2, V3, C3, V4, C4, V5, and C5) are denoted under the sequence. The Env-A244 peptides generated from CAT B, (blue line) CAT D (red line), CAT K (green line), CAT L (purple line), and CAT S (orange) are shown The solid and the dotted lines above the sequence represent the peptide fragments identified at the 90 min and the 16 hrs time point, respectively. These fragments could contain potential MHC class I and class II epitopes. The solid black lines below the sequence represent MHC class I epitopes, while the dashed black lines represent MHC class II epitopes as reported in the Las Alamos database.
Mentions: The peptides derived from CAT B, D, K, L, and S degradation of Env-A244 at 90 min (solid lines above the Env-A244 sequence) and additional peptides generated at 16 hrs (dotted lines above the Env-A244 sequence) were identified by mass spectrometry (Figure 3). The MHC class I and class II epitopes for Env-A244 as reported in the Los Alamos database are shown respectively beneath the amino acid sequence as a solid or hatched black line. Each of the cathepsin Env-A244 degradations generated clusters of potential peptide MHC class I and class II precursor epitopes (Figure 3 and Table 1) with dense clusters found in the C1, C2, V3, C3, C4, and C5 regions of Env-A244 that contained the CD4, chemokine, and majority of the neutralizing antibody binding sites. Each of the CAT generated varying number of peptides. The number of peptides produced was relatively consistent at 90 min and at 16 hrs with the exception of CAT B and D (Table 1). The concentration of individual peptides derived from each of the CAT degradations of Env-A244 was not assessed. Therefore, there could be varying concentrations of antigenic peptides in each of the CAT degradations.

Bottom Line: The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively.Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters.The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4(+) T-cell lines-derived from RV144 vaccinees.

View Article: PubMed Central - PubMed

Affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America.

ABSTRACT

Background: Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response.

Methods: In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4(+) T-cell lines derived from RV144 volunteers.

Results: Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4(+) T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial.

Conclusions: Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4(+)T cell and antibody responses in the RV144 vaccinees.

Show MeSH
Related in: MedlinePlus