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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

Proteasomes are unable to cleave HIV-1 Env proteins.Env-A244 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (panel A, lanes C) were treated with purified proteasomes isolated from activated CD4+ T-cells in the absence (lanes, P) or presence of epoxomicin, an irreversible proteasome inhibitor (lanes PI). Proteasomes were unable to cleave the Env proteins from two different HIV-1 clades. In panel B, RNaseB, a folded-glycosylated protein (lane C) was treated as in panel A with purified proteasomes in the absence (lane P) or presence of epoxomicin (lane PI). The proteasomes were also unable to cleave RNaseB. The functional activity and the specificity of the proteasomes were demonstrated by the proteolytic cleavage of Gag-p24 (panel C, lane C) in the absence (lane P) or presence of epoxomicin (lane PI). Env-A244 and RNaseB (panels D and E, lanes C) were subjected to the same treatment as in panels A and B. Env-A244 and RNaseB were treated with Endo-F and Endo-H followed by incubation with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Proteasomes were unable to cleave Env-A244 and RNaseB following treatment with either Endo-F or Endo-H. Env-A244 and RNaseB (panel F, lanes C) were reduced and alkylated (IAA) by treatment with iodioacetamine and DTT and then treated with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Env-A244 remained resistant while RNaseB became susceptible to proteasomal cleavage. Samples were run on a 4–20% gradient Tris-glycine polyacrylamide gel. The molecular weight markers are shown in lane M in panels A, B, D, E, and F.
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pone-0042579-g001: Proteasomes are unable to cleave HIV-1 Env proteins.Env-A244 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (panel A, lanes C) were treated with purified proteasomes isolated from activated CD4+ T-cells in the absence (lanes, P) or presence of epoxomicin, an irreversible proteasome inhibitor (lanes PI). Proteasomes were unable to cleave the Env proteins from two different HIV-1 clades. In panel B, RNaseB, a folded-glycosylated protein (lane C) was treated as in panel A with purified proteasomes in the absence (lane P) or presence of epoxomicin (lane PI). The proteasomes were also unable to cleave RNaseB. The functional activity and the specificity of the proteasomes were demonstrated by the proteolytic cleavage of Gag-p24 (panel C, lane C) in the absence (lane P) or presence of epoxomicin (lane PI). Env-A244 and RNaseB (panels D and E, lanes C) were subjected to the same treatment as in panels A and B. Env-A244 and RNaseB were treated with Endo-F and Endo-H followed by incubation with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Proteasomes were unable to cleave Env-A244 and RNaseB following treatment with either Endo-F or Endo-H. Env-A244 and RNaseB (panel F, lanes C) were reduced and alkylated (IAA) by treatment with iodioacetamine and DTT and then treated with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Env-A244 remained resistant while RNaseB became susceptible to proteasomal cleavage. Samples were run on a 4–20% gradient Tris-glycine polyacrylamide gel. The molecular weight markers are shown in lane M in panels A, B, D, E, and F.

Mentions: HIV-1 Env-A244 gp120 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (Figure 1 A, lanes C) were treated with proteasomes (lanes P) purified from activated CD4+ T-cells in the absence or presence of proteasome inhibitor (lanes PI), epoxomycin and analyzed on a gradient SDS-polyacrylamide gel. Env-A244 is heavily glycosylated with mannose being one of the principal sugar moieties. Although it has been reported that proteasomes are capable of cleaving glycoproteins [27], [28] proteasomes however, were unable to cleave the glycosylated and folded Env-A244 and gp140 proteins (Figure 1 A). Interestingly, Env-A244 has a gD-tag, and this was effectively cleaved by the proteasomes as identified by mass spectrometry (data not shown). It could be hypothesized that the presence of mannose residues and the folded structure could afford Env-A244 and gp140 a certain degree of protection from proteolytic degradation. This hypothesis is supported by previously published results demonstrating that proteasomes were unable to cleave RNaseB in its native glycosylated and folded form [28]. In our studies also, RNaseB (Figure 1 B, lane C) could not be cleaved by proteasomes (lane P). Again, proteasomes treated with epoxomicin served as a control (lane PI). The lack of RNaseB cleavage by proteasomes was not due to a loss of enzymatic activity as demonstrated by the cleavage of clade B Gag-p24 (Figure 1 C). The specificity of the proteasomal degradation (lane P) of Gag-p24 (lane C) was demonstrated by the lack of proteolytic degradation of Gag-p24 in the presence of the epoxomicin (lane PI).


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)

Proteasomes are unable to cleave HIV-1 Env proteins.Env-A244 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (panel A, lanes C) were treated with purified proteasomes isolated from activated CD4+ T-cells in the absence (lanes, P) or presence of epoxomicin, an irreversible proteasome inhibitor (lanes PI). Proteasomes were unable to cleave the Env proteins from two different HIV-1 clades. In panel B, RNaseB, a folded-glycosylated protein (lane C) was treated as in panel A with purified proteasomes in the absence (lane P) or presence of epoxomicin (lane PI). The proteasomes were also unable to cleave RNaseB. The functional activity and the specificity of the proteasomes were demonstrated by the proteolytic cleavage of Gag-p24 (panel C, lane C) in the absence (lane P) or presence of epoxomicin (lane PI). Env-A244 and RNaseB (panels D and E, lanes C) were subjected to the same treatment as in panels A and B. Env-A244 and RNaseB were treated with Endo-F and Endo-H followed by incubation with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Proteasomes were unable to cleave Env-A244 and RNaseB following treatment with either Endo-F or Endo-H. Env-A244 and RNaseB (panel F, lanes C) were reduced and alkylated (IAA) by treatment with iodioacetamine and DTT and then treated with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Env-A244 remained resistant while RNaseB became susceptible to proteasomal cleavage. Samples were run on a 4–20% gradient Tris-glycine polyacrylamide gel. The molecular weight markers are shown in lane M in panels A, B, D, E, and F.
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Related In: Results  -  Collection

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pone-0042579-g001: Proteasomes are unable to cleave HIV-1 Env proteins.Env-A244 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (panel A, lanes C) were treated with purified proteasomes isolated from activated CD4+ T-cells in the absence (lanes, P) or presence of epoxomicin, an irreversible proteasome inhibitor (lanes PI). Proteasomes were unable to cleave the Env proteins from two different HIV-1 clades. In panel B, RNaseB, a folded-glycosylated protein (lane C) was treated as in panel A with purified proteasomes in the absence (lane P) or presence of epoxomicin (lane PI). The proteasomes were also unable to cleave RNaseB. The functional activity and the specificity of the proteasomes were demonstrated by the proteolytic cleavage of Gag-p24 (panel C, lane C) in the absence (lane P) or presence of epoxomicin (lane PI). Env-A244 and RNaseB (panels D and E, lanes C) were subjected to the same treatment as in panels A and B. Env-A244 and RNaseB were treated with Endo-F and Endo-H followed by incubation with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Proteasomes were unable to cleave Env-A244 and RNaseB following treatment with either Endo-F or Endo-H. Env-A244 and RNaseB (panel F, lanes C) were reduced and alkylated (IAA) by treatment with iodioacetamine and DTT and then treated with purified proteasomes in the absence (lanes P) or presence of epoxomicin (lanes PI). Env-A244 remained resistant while RNaseB became susceptible to proteasomal cleavage. Samples were run on a 4–20% gradient Tris-glycine polyacrylamide gel. The molecular weight markers are shown in lane M in panels A, B, D, E, and F.
Mentions: HIV-1 Env-A244 gp120 (CRF01_AE) derived from CHO cells and gp140 (clade B) derived from H9 cells (Figure 1 A, lanes C) were treated with proteasomes (lanes P) purified from activated CD4+ T-cells in the absence or presence of proteasome inhibitor (lanes PI), epoxomycin and analyzed on a gradient SDS-polyacrylamide gel. Env-A244 is heavily glycosylated with mannose being one of the principal sugar moieties. Although it has been reported that proteasomes are capable of cleaving glycoproteins [27], [28] proteasomes however, were unable to cleave the glycosylated and folded Env-A244 and gp140 proteins (Figure 1 A). Interestingly, Env-A244 has a gD-tag, and this was effectively cleaved by the proteasomes as identified by mass spectrometry (data not shown). It could be hypothesized that the presence of mannose residues and the folded structure could afford Env-A244 and gp140 a certain degree of protection from proteolytic degradation. This hypothesis is supported by previously published results demonstrating that proteasomes were unable to cleave RNaseB in its native glycosylated and folded form [28]. In our studies also, RNaseB (Figure 1 B, lane C) could not be cleaved by proteasomes (lane P). Again, proteasomes treated with epoxomicin served as a control (lane PI). The lack of RNaseB cleavage by proteasomes was not due to a loss of enzymatic activity as demonstrated by the cleavage of clade B Gag-p24 (Figure 1 C). The specificity of the proteasomal degradation (lane P) of Gag-p24 (lane C) was demonstrated by the lack of proteolytic degradation of Gag-p24 in the presence of the epoxomicin (lane PI).

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