Mechanisms of HIV Protein Degradation into Epitopes: Implications for Vaccine Design

The degradation of HIV-derived proteins into epitopes displayed by MHC-I or MHC-II are the first events leading to the priming of HIV-specific immune responses and to the recognition of infected cells. Despite a wealth of information about peptidases involved in protein degradation, our knowledge of epitope presentation during HIV infection remains limited. Here we review current data on HIV protein degradation linking epitope production and immunodominance, viral evolution and impaired epitope presentation. We propose that an in-depth understanding of HIV antigen processing and presentation in relevant primary cells could be exploited to identify signatures leading to efficient or inefficient epitope presentation in HIV proteomes, and to improve the design of immunogens eliciting immune responses efficiently recognizing all infected cells

Variable Processing and Cross-presentation of HIV by Dendritic Cells and Macrophages Shapes CTL Immunodominance and Immune Escape

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8+ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation

Effet des antirétroviraux sur la voie d’apprêtement des antigènes et la présentation directe ainsi que croisée des épitopes par les CMH-I

Antigen processing by intracellular proteases and peptidases and epitope presentation are critical for recognition of pathogen-infected cells by CD8+ T lymphocytes. Here we show that several HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate proteasome and aminopeptidase activities involved in endogenous antigen presentation and cathepsin activities involved in antigen cross-presentation. Two HIV PIs acted directly on cathepsins and on their regulators by inhibiting kinases, NOX2 and the regulation of phagolysosomal pH, subsequently enhancing cathepsin activities. HIV PIs modified HIV protein degradation and epitope production in a sequence- and cell-dependent manner, altered direct- and cross-presentation and T cell-mediated killing, and partly changed the self-peptidome of primary cells. Drug-induced modulation of antigen processing and peptidome may provide an alternate therapeutic approach to modulate immune recognition.L’apprêtement antigénique par les protéases intracellulaires et la présentation des épitopes sont essentiels pour la reconnaissance des cellules infectées par les lymphocytes CD8+. Ici nous avons montré que certains inhibiteurs de la protéase de la VIH (IPs) modulent l’activité de la protéasome et aminopeptidase impliqué dans l’apprêtement antigénique endogène et l’activité cathepsins importante dans l’apprêtement croisée. Deux IPs agissent directement sur les cathepsins et leurs régulateurs en inhibant les activités kinase, NOX2 et en régulant le pH phagolysosomal. Les IPs ont changé la dégradation des protéines viral et la production des épitopes de façon séquence- et cellule-spécifique, ont altéré la présentation direct et croisée des épitopes, et ont partiellement changé l’auto-peptidome des cellules primaires. La modulation par les drogues de l’apprêtement et la présentation des épitopes peut fournir une approche thérapeutique alternative pour moduler la reconnaissance immunitaire

Slow degradation of TW10-containing peptides results in high amounts of B57-TW10 available for cross-presentation.

<p>A. Two nmol of p24–31mer (GSDIAGTTSTLQEQIGWMTNNPPIPVGGEIY, aa 101–131 in Gag p24) were degraded in 15μg of whole cell extracts from immature and mature DCs and Møs for 10, 30, 60, and 120 minutes in degradation buffer at pH7.4, pH5.5 or pH4.0. Degradation products identified by mass spectrometry were grouped according to their lengths of fragments as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004725#ppat.1004725.g003" target="_blank">Fig. 3</a>. The contribution of each category of peptides to the total intensity of all degradation products is shown at each time point. B. All degradation products of p24–31mer were grouped into fragments containing B57-TW10 (black), B57-TW10 epitope with N-terminal extensions (white), B57-TW10 epitope with C-terminal extensions (red), B57-TW10 epitope with N- and C-terminal extensions (green), antitopes defined as fragments lacking B57-TW10 (blue), or the original peptide (gray), respectively. The contribution of each category of peptides to the total intensity of all degradation products is shown at each time point.</p

The immunodominant epitope B57-KF11 is more efficiently produced in cross-presentation-competent compartments than subdominant epitope B57-ISW9.

<p>A. Two nmol of p24–35mer (MVHQAISPRTLNAWVKVVEEKAFSPEVIPMFAALS, aa 10–44 in Gag p24) were degraded in 15μg of whole cell extracts from immature and mature DCs and Møs for 10, 30, 60, and 120 minutes in degradation buffer at pH7.4, pH5.5 or pH4.0. Degradation products identified by mass spectrometry were grouped according to their lengths of fragments: equal or longer than 26 aa (blue), 19–25 aa (orange), 13–18 aa (gray), 8–12 aa (red), and fragments equal or shorter than 7 aa (light gray). The peak area of each identified peptide was calculated with Proteome Discoverer and the contribution of each category of peptides to the total intensity of all degradation products is shown at each time point. B. All degradation products of p24–35mer were identified as described in (A). Peptides were grouped into fragments containing B57-ISW9 and B57-KF11 epitopes (black), containing only B57-KF11 epitope (red), containing only B57-ISW9 epitope (blue), or neither epitope (gray), respectively. C. Cleavage patterns of p24–35mer incubated with whole cell extracts from immature DCs for 30 minutes (upper panel) or 120 minutes (lower panel) at pH7.4, pH5.5, and pH4.0 are shown as the contribution of each cleavage site, presented as cleavage N-terminal or C-terminal to a specific amino acid, to the total intensity of all degradation products. For (A-C) data are representative of three independent experiments with three different donors.</p

Limited degradation of RK9-containing fragments results in cross-presentation of high amounts of HLA-A03 RK9 epitope.

<p>A. Immature DCs (◇) or Møs (◆) were incubated with recombinant HIV-1 p55 protein and used as antigen presenting cells in an overnight IFN-gamma ELISPOT assay with HLA-A03 RK9-specific CTLs as effector cells. CTL responses in form of spot forming cells are shown for n≥5 donors. B. CTL responses to immature DCs (◇) and immature Møs (◆) pulsed with different concentrations of optimal epitope A03-RK9 were measured as described before. C. Immature DCs (open bars) or Møs (solid bars) were incubated with different concentrations of recombinant HIV-1 p55 protein and used as antigen presenting cells in an overnight IFN-gamma ELISPOT assay with HLA-A03 RK9-specific CTLs as effector cells. CTL responses in form of spot forming cells are shown. D. Cleavage patterns of p17–17mer incubated with whole cell extracts from immature DCs for 120 minutes at pH7.4, pH5.5, and pH4.0 are shown as the contribution of each cleavage site, presented as cleavage N-terminal or C-terminal to a specific amino acid, to the total intensity of all degradation products. For (B-D) data are representative of two independent experiments with different donors.</p

The immunodominant HLA-B57-restricted TW10 and KF11 epitopes are more efficiently cross-presented than subdominant ISW9 epitope.

<p>A. Immature DCs (open symbols) and Møs (solid symbols) were incubated with recombinant HIV-1 p24 protein and used as antigen presenting cells in an overnight IFN-gamma ELISPOT assay with HLA-B57 ISW9-specific (☐), KF11-specific (○), and TW10-specific (Δ) CTL clones as effector cells. CTL responses in form of spot forming cells are shown for n≥10 donors. B. CTL responses to immature DCs (open symbols) and Møs (solid symbols) pulsed with different concentrations of optimal epitope B57-ISW9 (☐) or B57-TW10 (Δ) were measured in an overnight IFN-gamma ELISPOT assay. Data are representative of two independent experiments with different donors. Due to the limited number of DCs and Møs generated from HLA-B57⁺ donors, the titration of epitopes was only done for B57-ISW9 and B57-TW10. C. Immature DCs (open bars) and immature Møs (solid bars) were pre-treated with a cocktail of protease inhibitors to reduce intracellular degradation before addition of different concentrations of recombinant HIV-1 p24 protein at 37°C or 4°C. Internalized p24 protein was determined by a standard p24 ELISA assay using whole cell extracts from lysed DCs and Møs. Results are from three independent experiments with different donors and show mean ± SD. D. Immature DCs (open bars) or immature Møs (solid bars) were incubated with different concentrations of recombinant HIV-1 p24 protein and used as antigen presenting cells to B57-KF11-specific CTLs. Responses in form of spot forming cells are shown. Data are representative of two independent experiments with different donors.</p