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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 are able to cleave HIV-1 Env-A244 and CAT degradation products are susceptible to proteasomal degradation.HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient Tris-glycine polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.
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pone-0042579-g002: CAT are able to cleave HIV-1 Env-A244 and CAT degradation products are susceptible to proteasomal degradation.HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient Tris-glycine polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.

Mentions: Env-A244 was subjected to cathepsin (CAT) degradation (Figures 2 A and 2 B) and then the degradation products were analyzed on a gradient SDS-polyacrylamide gel. The 90 min cathepsin degradation products were then subjected to proteasomal degradation for 16 hrs (Figures 2 C and 2 D). Although Env-A244 (Figures 2 A and 2 B, lanes 0) was resistant to proteasomal degradation (Figure 1 A), the protein was susceptible to CAT B, D, K, L, and S degradation (Figures 2 A and 2 B). The 90 min (lanes 90) and 16 hrs degradation products (lanes O/N) are respectively shown in Figures 2 A and 2 B. The respective CAT degradation products at 90 min were then subjected to proteasomal degradation in the presence (Figures 2 C and 2 D, lanes PI) or absence (lanes P) of epoxomicin. Env A-244 was resistant to cathepsin H degradation at the 90 min as well as at the 16 hrs time point (Figure 2A, lanes 90 and O/N), and as a result remained resistant to proteasomal cleavage in the presence or absence of epoxomicin (Figure 2 C, lanes PI and P, respectively).


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 are able to cleave HIV-1 Env-A244 and CAT degradation products are susceptible to proteasomal degradation.HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient Tris-glycine polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042579-g002: CAT are able to cleave HIV-1 Env-A244 and CAT degradation products are susceptible to proteasomal degradation.HIV-1 Env-A244 (panels A and B, lanes 0) was treated with CAT for 90 min or 16 hrs and then analyzed on a 4–20% gradient Tris-glycine polyacrylamide gel. The 90 min (lanes 90) and 16 hrs (lanes 0/N) degradation pattern with the various CAT are shown in panels A and B. Env-A244 was treated with the individual CAT for 90 min followed by proteasomes in the presence (panels C and D, lanes PI) or absence of epoxomicin (lanes P). The molecular weight markers are shown in lane M in all panels.
Mentions: Env-A244 was subjected to cathepsin (CAT) degradation (Figures 2 A and 2 B) and then the degradation products were analyzed on a gradient SDS-polyacrylamide gel. The 90 min cathepsin degradation products were then subjected to proteasomal degradation for 16 hrs (Figures 2 C and 2 D). Although Env-A244 (Figures 2 A and 2 B, lanes 0) was resistant to proteasomal degradation (Figure 1 A), the protein was susceptible to CAT B, D, K, L, and S degradation (Figures 2 A and 2 B). The 90 min (lanes 90) and 16 hrs degradation products (lanes O/N) are respectively shown in Figures 2 A and 2 B. The respective CAT degradation products at 90 min were then subjected to proteasomal degradation in the presence (Figures 2 C and 2 D, lanes PI) or absence (lanes P) of epoxomicin. Env A-244 was resistant to cathepsin H degradation at the 90 min as well as at the 16 hrs time point (Figure 2A, lanes 90 and O/N), and as a result remained resistant to proteasomal cleavage in the presence or absence of epoxomicin (Figure 2 C, lanes PI and P, respectively).

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