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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

A schematic representation of the differential processing of the components of the RV144 vaccine.The RV144 vaccine consists of canarypox vector ALVAC (vCP1521), carrying HIV clade B and circulating recombinant form (CRF)01_AE gag, pro and env genes, and AIDSVAX®B/E genetically engineered gp120 protein from viruses of subtypes B and CRF01_AE. After entry into the cell, the ALVAC canary pox vector will generate protein antigens in the cytosol after transcription and translation. These proteins are then processed through the classical MHC class I pathway involving the proteasomal complex. The phagocytosed AIDSVAX®B/E protein antigens will enter the endosome/lysosome compartment and will be cleaved by the CAT. Some of the peptides generated within the endosome/lysosome compartment can be retrotranslocated into the cytosol and enter the classical MHC class I pathway. The peptides containing the MHC class II epiotpes enter the MIIC compartment from the endosome/lysosome where they are then loaded onto class II molecules and transported to the cell surface.
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pone-0042579-g010: A schematic representation of the differential processing of the components of the RV144 vaccine.The RV144 vaccine consists of canarypox vector ALVAC (vCP1521), carrying HIV clade B and circulating recombinant form (CRF)01_AE gag, pro and env genes, and AIDSVAX®B/E genetically engineered gp120 protein from viruses of subtypes B and CRF01_AE. After entry into the cell, the ALVAC canary pox vector will generate protein antigens in the cytosol after transcription and translation. These proteins are then processed through the classical MHC class I pathway involving the proteasomal complex. The phagocytosed AIDSVAX®B/E protein antigens will enter the endosome/lysosome compartment and will be cleaved by the CAT. Some of the peptides generated within the endosome/lysosome compartment can be retrotranslocated into the cytosol and enter the classical MHC class I pathway. The peptides containing the MHC class II epiotpes enter the MIIC compartment from the endosome/lysosome where they are then loaded onto class II molecules and transported to the cell surface.

Mentions: The RV144 phase III trial vaccine regimen consisted of four doses of a recombinant canary pox vector-based priming immunogen, ALVAC (vCP1521) administered at 0, 4, 12, and 24 weeks, and two doses of AIDSVAX®B/E co-administered at 12 and 24 weeks. AIDSVAX®B/E consists of genetically engineered HIV-1 gp120 proteins from viruses of subtypes B and CRF01_AE. We hypothesize that each component of the vaccine might be processed and presented in a different manner, and this is schematically represented in Figure 10. Theoretically, the ALVAC canary pox vector will generate protein antigens in the cytosol after transcription and translation, while the AIDSVAX®B/E protein antigens will be phagocytosed into endosome/lysosome compartment. Normally, Gag is proteolytically cleaved by the proteasome and follows the classical MHC class I pathway. Since HIV-1 Env was resistant to proteasomal degradation (Figure 1 A and Table 2) we hypothesize that the Env within the cytosol could potentially be processed by a mechanism known as autophagy (phagocytosis within the cell) wherein antigens are taken up into endosomes and cleaved by CAT. These peptides can either be directly loaded onto MHC class II molecules or retrotranslocated into the cytosol where they can then enter the proteasome/classical MHC class I pathway. Our results show that the potential Env-A244 MHC class I epitopes are destroyed to a greater extent as a result of proteasomal cleavage following CAT digestion (Table 2). This could limit the relative amounts of peptides available for presentation by the classical MHC class I pathway and may be a further possible explanation for the poor Env-specific CD8+ T-cell response seen in the RV144 Phase III trial. Although, other studies have demonstrated a strong and broad Gag-specific CD8+ T-cell response [48]–[50] the weak Gag response seen in the RV144 phase III trial is perplexing.


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)

A schematic representation of the differential processing of the components of the RV144 vaccine.The RV144 vaccine consists of canarypox vector ALVAC (vCP1521), carrying HIV clade B and circulating recombinant form (CRF)01_AE gag, pro and env genes, and AIDSVAX®B/E genetically engineered gp120 protein from viruses of subtypes B and CRF01_AE. After entry into the cell, the ALVAC canary pox vector will generate protein antigens in the cytosol after transcription and translation. These proteins are then processed through the classical MHC class I pathway involving the proteasomal complex. The phagocytosed AIDSVAX®B/E protein antigens will enter the endosome/lysosome compartment and will be cleaved by the CAT. Some of the peptides generated within the endosome/lysosome compartment can be retrotranslocated into the cytosol and enter the classical MHC class I pathway. The peptides containing the MHC class II epiotpes enter the MIIC compartment from the endosome/lysosome where they are then loaded onto class II molecules and transported to the cell surface.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412807&req=5

pone-0042579-g010: A schematic representation of the differential processing of the components of the RV144 vaccine.The RV144 vaccine consists of canarypox vector ALVAC (vCP1521), carrying HIV clade B and circulating recombinant form (CRF)01_AE gag, pro and env genes, and AIDSVAX®B/E genetically engineered gp120 protein from viruses of subtypes B and CRF01_AE. After entry into the cell, the ALVAC canary pox vector will generate protein antigens in the cytosol after transcription and translation. These proteins are then processed through the classical MHC class I pathway involving the proteasomal complex. The phagocytosed AIDSVAX®B/E protein antigens will enter the endosome/lysosome compartment and will be cleaved by the CAT. Some of the peptides generated within the endosome/lysosome compartment can be retrotranslocated into the cytosol and enter the classical MHC class I pathway. The peptides containing the MHC class II epiotpes enter the MIIC compartment from the endosome/lysosome where they are then loaded onto class II molecules and transported to the cell surface.
Mentions: The RV144 phase III trial vaccine regimen consisted of four doses of a recombinant canary pox vector-based priming immunogen, ALVAC (vCP1521) administered at 0, 4, 12, and 24 weeks, and two doses of AIDSVAX®B/E co-administered at 12 and 24 weeks. AIDSVAX®B/E consists of genetically engineered HIV-1 gp120 proteins from viruses of subtypes B and CRF01_AE. We hypothesize that each component of the vaccine might be processed and presented in a different manner, and this is schematically represented in Figure 10. Theoretically, the ALVAC canary pox vector will generate protein antigens in the cytosol after transcription and translation, while the AIDSVAX®B/E protein antigens will be phagocytosed into endosome/lysosome compartment. Normally, Gag is proteolytically cleaved by the proteasome and follows the classical MHC class I pathway. Since HIV-1 Env was resistant to proteasomal degradation (Figure 1 A and Table 2) we hypothesize that the Env within the cytosol could potentially be processed by a mechanism known as autophagy (phagocytosis within the cell) wherein antigens are taken up into endosomes and cleaved by CAT. These peptides can either be directly loaded onto MHC class II molecules or retrotranslocated into the cytosol where they can then enter the proteasome/classical MHC class I pathway. Our results show that the potential Env-A244 MHC class I epitopes are destroyed to a greater extent as a result of proteasomal cleavage following CAT digestion (Table 2). This could limit the relative amounts of peptides available for presentation by the classical MHC class I pathway and may be a further possible explanation for the poor Env-specific CD8+ T-cell response seen in the RV144 Phase III trial. Although, other studies have demonstrated a strong and broad Gag-specific CD8+ T-cell response [48]–[50] the weak Gag response seen in the RV144 phase III trial is perplexing.

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