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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

Env-A244 peptides derived by CAT D, K, and L cleavage induce a poly-functional cytokine response from antigen-specific CD4+ T-cell lines.HIV-1 Env specific CD4+ T cell lines derived from two volunteers from the RV144 phase III trial (144620 and 144193) were stimulated with either the CMDR Env peptides, SEB, peptides generated from Env-A244 cleaved by CAT D, CAT K, or CAT L, or cultured in media only. The CD4+ T cells were analyzed for the generation of CD107a (red), IFN-γ (brown), IL-2 (light green), MIP1β (light blue) and TNF-α (purple) by flow cytometry. The pie charts (panels A and B) demonstrate the fraction of cells with one (green), two (red), three (purple), four (orange), or five (light blue) cytokines generated from the same cell, and the circular lines arching the pie chart represent the five cytokines/chemokines examined. The data is denoted in a graphical format in panels C and D as a percentage of CD4+ T-cells capable of generating each of the 31 possible cytokine combinations. The color designations for each of the experimental conditions shown in panels C and D are as follows: unstimulated (blue bar); SEB (yellow bar); CMDR peptides (red bar); CAT-D Env-A244 derived peptides (green bar); CAT-K Env-A244 derived peptides (orange bar); and CAT-L Env-A244 derived peptides (purple bar).
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pone-0042579-g009: Env-A244 peptides derived by CAT D, K, and L cleavage induce a poly-functional cytokine response from antigen-specific CD4+ T-cell lines.HIV-1 Env specific CD4+ T cell lines derived from two volunteers from the RV144 phase III trial (144620 and 144193) were stimulated with either the CMDR Env peptides, SEB, peptides generated from Env-A244 cleaved by CAT D, CAT K, or CAT L, or cultured in media only. The CD4+ T cells were analyzed for the generation of CD107a (red), IFN-γ (brown), IL-2 (light green), MIP1β (light blue) and TNF-α (purple) by flow cytometry. The pie charts (panels A and B) demonstrate the fraction of cells with one (green), two (red), three (purple), four (orange), or five (light blue) cytokines generated from the same cell, and the circular lines arching the pie chart represent the five cytokines/chemokines examined. The data is denoted in a graphical format in panels C and D as a percentage of CD4+ T-cells capable of generating each of the 31 possible cytokine combinations. The color designations for each of the experimental conditions shown in panels C and D are as follows: unstimulated (blue bar); SEB (yellow bar); CMDR peptides (red bar); CAT-D Env-A244 derived peptides (green bar); CAT-K Env-A244 derived peptides (orange bar); and CAT-L Env-A244 derived peptides (purple bar).

Mentions: The functional relevance of peptides derived from degradation of Env-A244 by CAT D, K, and L were evaluated using either peripheral blood mononuclear cells (PBMCs) obtained from RV144 volunteers (Figure 7) or from CD4+ T-cell lines derived from RV144 volunteers (Figures 8 and 9). The number of IFN-γ producing cells was determined by an ELISPOT assay, using PBMCs from the peak of immunogenicity at the 2-week time point following the completion of the RV144 immunization schedule. The Env-A244 peptide repertoire derived from degradation by CAT D and K were examined individually for their potential to induce IFN-γ. Although the ELISPOT responses were weak, the peptides generated from CAT K induced IFN-γ in 2 out of the 10 vaccinees tested (Figure 7, bottom panel) that were at least twice the background (buffer control). The ELISPOT well images from the two positive responders stimulated with peptides derived from Env-A244 CAT D and CAT K digests including images of wells stimulated with media, PHA, CMV peptide pool, and CAT buffers are shown in Figures S1 and S2. Although the ELISPOT responses are low, the results are consistent with a recent study analyzing the Env-specific IFN-γresponses generated from PBMCs of RV144 volunteers. Furthermore, depletion studies indicated that the IFN-γgeneration was from CD4+ T cells [38].


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)

Env-A244 peptides derived by CAT D, K, and L cleavage induce a poly-functional cytokine response from antigen-specific CD4+ T-cell lines.HIV-1 Env specific CD4+ T cell lines derived from two volunteers from the RV144 phase III trial (144620 and 144193) were stimulated with either the CMDR Env peptides, SEB, peptides generated from Env-A244 cleaved by CAT D, CAT K, or CAT L, or cultured in media only. The CD4+ T cells were analyzed for the generation of CD107a (red), IFN-γ (brown), IL-2 (light green), MIP1β (light blue) and TNF-α (purple) by flow cytometry. The pie charts (panels A and B) demonstrate the fraction of cells with one (green), two (red), three (purple), four (orange), or five (light blue) cytokines generated from the same cell, and the circular lines arching the pie chart represent the five cytokines/chemokines examined. The data is denoted in a graphical format in panels C and D as a percentage of CD4+ T-cells capable of generating each of the 31 possible cytokine combinations. The color designations for each of the experimental conditions shown in panels C and D are as follows: unstimulated (blue bar); SEB (yellow bar); CMDR peptides (red bar); CAT-D Env-A244 derived peptides (green bar); CAT-K Env-A244 derived peptides (orange bar); and CAT-L Env-A244 derived peptides (purple bar).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412807&req=5

pone-0042579-g009: Env-A244 peptides derived by CAT D, K, and L cleavage induce a poly-functional cytokine response from antigen-specific CD4+ T-cell lines.HIV-1 Env specific CD4+ T cell lines derived from two volunteers from the RV144 phase III trial (144620 and 144193) were stimulated with either the CMDR Env peptides, SEB, peptides generated from Env-A244 cleaved by CAT D, CAT K, or CAT L, or cultured in media only. The CD4+ T cells were analyzed for the generation of CD107a (red), IFN-γ (brown), IL-2 (light green), MIP1β (light blue) and TNF-α (purple) by flow cytometry. The pie charts (panels A and B) demonstrate the fraction of cells with one (green), two (red), three (purple), four (orange), or five (light blue) cytokines generated from the same cell, and the circular lines arching the pie chart represent the five cytokines/chemokines examined. The data is denoted in a graphical format in panels C and D as a percentage of CD4+ T-cells capable of generating each of the 31 possible cytokine combinations. The color designations for each of the experimental conditions shown in panels C and D are as follows: unstimulated (blue bar); SEB (yellow bar); CMDR peptides (red bar); CAT-D Env-A244 derived peptides (green bar); CAT-K Env-A244 derived peptides (orange bar); and CAT-L Env-A244 derived peptides (purple bar).
Mentions: The functional relevance of peptides derived from degradation of Env-A244 by CAT D, K, and L were evaluated using either peripheral blood mononuclear cells (PBMCs) obtained from RV144 volunteers (Figure 7) or from CD4+ T-cell lines derived from RV144 volunteers (Figures 8 and 9). The number of IFN-γ producing cells was determined by an ELISPOT assay, using PBMCs from the peak of immunogenicity at the 2-week time point following the completion of the RV144 immunization schedule. The Env-A244 peptide repertoire derived from degradation by CAT D and K were examined individually for their potential to induce IFN-γ. Although the ELISPOT responses were weak, the peptides generated from CAT K induced IFN-γ in 2 out of the 10 vaccinees tested (Figure 7, bottom panel) that were at least twice the background (buffer control). The ELISPOT well images from the two positive responders stimulated with peptides derived from Env-A244 CAT D and CAT K digests including images of wells stimulated with media, PHA, CMV peptide pool, and CAT buffers are shown in Figures S1 and S2. Although the ELISPOT responses are low, the results are consistent with a recent study analyzing the Env-specific IFN-γresponses generated from PBMCs of RV144 volunteers. Furthermore, depletion studies indicated that the IFN-γgeneration was from CD4+ T cells [38].

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