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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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Proteasomal cleavage maps following CAT degradation of Env-A244.Env-A244 was subjected to CAT digestion for 90 min followed by proteasomal degradation for 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 additional Env-A244 peptides generated from proteasomal degradation of CAT B (blue line), CAT D (red line), CAT K (green line), CAT L (purple line), and CAT S (orange) digestions are shown. The lines above the sequence represent additional peptide fragments identified after proteasomal degradation. 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-g004: Proteasomal cleavage maps following CAT degradation of Env-A244.Env-A244 was subjected to CAT digestion for 90 min followed by proteasomal degradation for 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 additional Env-A244 peptides generated from proteasomal degradation of CAT B (blue line), CAT D (red line), CAT K (green line), CAT L (purple line), and CAT S (orange) digestions are shown. The lines above the sequence represent additional peptide fragments identified after proteasomal degradation. 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: Generally in a cell these CAT generated peptides can be retrotranslocated into the cytosol for further degradation by the proteasomes, therefore we incubated the cathepsin degradation products with purified proteasomes in vitro. When the 90 min CAT degradations were subjected to proteasomal degradation (Figure 4), in some cases, an alternative peptide repertoire was generated even though there was no apparent significant increase in the number of peptides identified by mass spectrometry with the exception of CAT L (Figure 4 and Table 1). The majority of the reported Env-A244 epitopes were present in the peptides generated by the cathepsin degradation (Table 2). Although proteasomal cleavage of the cathepsin degradation products destroyed approximately 50% of the peptides containing MHC class I epitopes, thus potentially removing the epitope for presentation through the alternative MHC class I pathway, it also generated 3 MHC class I epitopes that were previously absent in the peptides identified from CAT degradation (Table 2). The CAT generated peptides that contained known MHC class II epitopes were not affected by further treatment with proteasomes (Table 2).


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)

Proteasomal cleavage maps following CAT degradation of Env-A244.Env-A244 was subjected to CAT digestion for 90 min followed by proteasomal degradation for 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 additional Env-A244 peptides generated from proteasomal degradation of CAT B (blue line), CAT D (red line), CAT K (green line), CAT L (purple line), and CAT S (orange) digestions are shown. The lines above the sequence represent additional peptide fragments identified after proteasomal degradation. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412807&req=5

pone-0042579-g004: Proteasomal cleavage maps following CAT degradation of Env-A244.Env-A244 was subjected to CAT digestion for 90 min followed by proteasomal degradation for 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 additional Env-A244 peptides generated from proteasomal degradation of CAT B (blue line), CAT D (red line), CAT K (green line), CAT L (purple line), and CAT S (orange) digestions are shown. The lines above the sequence represent additional peptide fragments identified after proteasomal degradation. 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: Generally in a cell these CAT generated peptides can be retrotranslocated into the cytosol for further degradation by the proteasomes, therefore we incubated the cathepsin degradation products with purified proteasomes in vitro. When the 90 min CAT degradations were subjected to proteasomal degradation (Figure 4), in some cases, an alternative peptide repertoire was generated even though there was no apparent significant increase in the number of peptides identified by mass spectrometry with the exception of CAT L (Figure 4 and Table 1). The majority of the reported Env-A244 epitopes were present in the peptides generated by the cathepsin degradation (Table 2). Although proteasomal cleavage of the cathepsin degradation products destroyed approximately 50% of the peptides containing MHC class I epitopes, thus potentially removing the epitope for presentation through the alternative MHC class I pathway, it also generated 3 MHC class I epitopes that were previously absent in the peptides identified from CAT degradation (Table 2). The CAT generated peptides that contained known MHC class II epitopes were not affected by further treatment with proteasomes (Table 2).

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