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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

Biochemical characteristics of the CAT and CAT followed by proteasomal degradation products of Env-A244.The Env-A244 peptides that were identified in Figures 3 A and 3 B were plotted to compare the size and frequency of the peptides generated after individual CAT (panel A) or individual CAT followed by proteasomal degradation (panel B). The molecular weights (mol wt), and the isoelectric points (pI) of the peptides after each of the degradations were also analyzed (panels C, D). No significant differences in the amino acid length (panels A, B), pI or mol wt (panels C, D) of the peptides produced from the cleavage products of Env-A244 were observed.
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pone-0042579-g006: Biochemical characteristics of the CAT and CAT followed by proteasomal degradation products of Env-A244.The Env-A244 peptides that were identified in Figures 3 A and 3 B were plotted to compare the size and frequency of the peptides generated after individual CAT (panel A) or individual CAT followed by proteasomal degradation (panel B). The molecular weights (mol wt), and the isoelectric points (pI) of the peptides after each of the degradations were also analyzed (panels C, D). No significant differences in the amino acid length (panels A, B), pI or mol wt (panels C, D) of the peptides produced from the cleavage products of Env-A244 were observed.

Mentions: The minimum required length for peptides to bind to MHC class I and class II molecules are 8 and 12 amino acids, respectively. CAT B, D, K, L, and S generated peptides ranging from 5 amino acids to greater than 19 amino acids with no significant differences in the length of peptides between the different degradations (Figures 6 A and 6 B). The majority of the peptides generated by CAT (Figure 6 A) or CAT followed by proteasomes (Figure 6 B) had the requisite peptide length that could potentially bind to MHC class I molecules. However, only about a third of the generated epitopes had the requisite peptide length required for binding to the MHC class II molecules. The peptides generated by the various CAT (Figure 6 C) or various CAT followed by proteasomes (Figure 6 D) clustered into two isoelectric ranges (pH 4–6 and pH 8–10) with no significant differences in the molecular weight of the peptides (http://au.expasy.org).


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)

Biochemical characteristics of the CAT and CAT followed by proteasomal degradation products of Env-A244.The Env-A244 peptides that were identified in Figures 3 A and 3 B were plotted to compare the size and frequency of the peptides generated after individual CAT (panel A) or individual CAT followed by proteasomal degradation (panel B). The molecular weights (mol wt), and the isoelectric points (pI) of the peptides after each of the degradations were also analyzed (panels C, D). No significant differences in the amino acid length (panels A, B), pI or mol wt (panels C, D) of the peptides produced from the cleavage products of Env-A244 were observed.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042579-g006: Biochemical characteristics of the CAT and CAT followed by proteasomal degradation products of Env-A244.The Env-A244 peptides that were identified in Figures 3 A and 3 B were plotted to compare the size and frequency of the peptides generated after individual CAT (panel A) or individual CAT followed by proteasomal degradation (panel B). The molecular weights (mol wt), and the isoelectric points (pI) of the peptides after each of the degradations were also analyzed (panels C, D). No significant differences in the amino acid length (panels A, B), pI or mol wt (panels C, D) of the peptides produced from the cleavage products of Env-A244 were observed.
Mentions: The minimum required length for peptides to bind to MHC class I and class II molecules are 8 and 12 amino acids, respectively. CAT B, D, K, L, and S generated peptides ranging from 5 amino acids to greater than 19 amino acids with no significant differences in the length of peptides between the different degradations (Figures 6 A and 6 B). The majority of the peptides generated by CAT (Figure 6 A) or CAT followed by proteasomes (Figure 6 B) had the requisite peptide length that could potentially bind to MHC class I molecules. However, only about a third of the generated epitopes had the requisite peptide length required for binding to the MHC class II molecules. The peptides generated by the various CAT (Figure 6 C) or various CAT followed by proteasomes (Figure 6 D) clustered into two isoelectric ranges (pH 4–6 and pH 8–10) with no significant differences in the molecular weight of the peptides (http://au.expasy.org).

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