How to develop a Controlled Human Infection Model for Clostridioides difficile.

BACKGROUND: Clostridioides difficile (C. difficile) remains the leading cause of healthcare-associated diarrhoea, posing treatment challenges due to antibiotic resistance and high relapse rates. Fecal microbiota transplantation (FMT) is a novel treatment strategy to prevent relapses of C. difficile infection (CDI), however the exact components conferring colonisation resistance are unknown, hampering its translation to a medicinal product. Development of novel products independent of antibiotics, which increase colonisation resistance or induce protective immune mechanisms are urgently needed. OBJECTIVES: To establish a framework for a Controlled Human Infection Model (CHIM) for C. difficile, in which healthy volunteers are exposed to toxigenic C. difficile spores, offering the possibility to test novel approaches and identify microbiota and immunological targets. Whereas experimental exposure to non-toxigenic C. difficile (NTCD) has been done before, a toxigenic C. difficile CHIM faces ethical, scientific, logistical and biosafety challenges. SOURCES: Specific challenges in developing a C. difficile CHIM were discussed by a group of international experts during a workshop organized by Inno4Vac, an IHI-funded consortium. CONTENT: The experts agreed that the main challenges are: developing a clinically relevant CHIM which induces mild to moderate CDI symptoms but no severe CDI, determining optimal C. difficile inoculum dose and understanding the timing and duration of antibiotic pre-treatment in inducing susceptibility to CDI in healthy volunteers. IMPLICATIONS: Should these challenges be tackled, a C. difficile CHIM not only provides a way forward for the testing of novel products but also offers a framework for better understanding of the pathophysiology, pathogenesis and immunology of C. difficile colonisation and infection.

Regulatory workshop on challenge strain development and GMP manufacture - A stakeholder meeting report.

Within the Innovative Health Initiative (IHI) Inno4Vac CHIMICHURRI project, a regulatory workshop was organised on the development and manufacture of challenge agent strains for Controlled Human Infection Model (CHIM) studies. Developers are often uncertain about which GMP requirements or regulatory guidelines apply but should be guided by the 2022 technical white paper "Considerations on the Principles of Development and Manufacturing Qualities of Challenge Agents for Use in Human Infection Models" (published by hVIVO, Wellcome Trust, HIC-Vac consortium members). Where those recommendations cannot be met, regulators advise following the "Principles of GMP" until definitive guidelines are available. Sourcing wild-type virus isolates is a significant challenge for developers. Still, it is preferred over reverse genetics challenge strains for several reasons, including implications and regulations around genetically modified organisms (GMOs). Official informed consent guidelines for collecting isolates are needed, and the characterisation of these isolates still presents risks and uncertainty. Workshop topics included ethics, liability, standardised clinical endpoints, selection criteria, sharing of challenge agents, and addressing population heterogeneity concerning vaccine response and clinical course. The organisers are confident that the workshop discussions will contribute to advancing ethical, safe, and high-quality CHIM studies of influenza, RSV and C. difficile, including adequate regulatory frameworks.

Ethical approval for controlled human infectious model clinical trial protocols - A workshop report.

Controlled Human Infectious Model studies (CHIM) involve deliberately exposing volunteers to pathogens. To discuss ethical issues related to CHIM, the European Vaccine Initiative and the International Alliance for Biological Standardization organised the workshop "Ethical Approval for CHIM Clinical Trial Protocols", which took place on May 30-31, 2023, in Brussels, Belgium. The event allowed CHIM researchers, regulators, ethics committee (EC) members, and ethicists to examine the ethical criteria for CHIM and the role(s) of CHIM in pharmaceutical development. The discussions led to several recommendations, including continued assurance that routine ethical requirements are met, assurance that participants are well-informed, and that preparation of study documents must be both ethically and scientifically sound from an early stage. Study applications must clearly state the rationale for the challenge compared to alternative study designs. ECs need to have clear guidance and procedures for evaluating social value and assessing third-party risks. Among other things, public trust in research requires minimisation of harm to healthy volunteers and third-party risk. Other important considerations include appropriate stakeholder engagement, public education, and access to health care for participants after the study.

Innate Immune Responses to Chimpanzee Adenovirus Vector 155 Vaccination in Mice and Monkeys.

Replication-deficient chimpanzee adenovirus (ChAd) vectors represent an attractive vaccine platform and are thus employed as vaccine candidates against several infectious diseases. Since inducing effective immunity depends on the interplay between innate and adaptive immunity, a deeper understanding of innate immune responses elicited by intramuscularly injected ChAd vectors in tissues can advance the platform's development. Using different candidate vaccines based on the Group C ChAd type 155 (ChAd155) vector, we characterized early immune responses in injected muscles and draining lymph nodes (dLNs) from mice, and complemented these analyses by evaluating cytokine responses and gene expression patterns in peripheral blood from ChAd155-injected macaques. In mice, vector DNA levels gradually decreased post-immunization, but local transgene mRNA expression exhibited two transient peaks [at 6 h and Day (D)5], which were most obvious in dLNs. This dynamic pattern was mirrored by the innate responses in tissues, which developed as early as 1-3 h (cytokines/chemokines) or D1 (immune cells) post-vaccination. They were characterized by a CCL2- and CXCL9/10-dominated chemokine profile, peaking at 6 h (with CXCL10/CCL2 signals also detectable in serum) and D7, and clear immune-cell infiltration peaks at D1/D2 and D6/D7. Experiments with a green fluorescent protein-expressing ChAd155 vector revealed infiltrating hematopoietic cell subsets at the injection site. Cell infiltrates comprised mostly monocytes in muscles, and NK cells, T cells, dendritic cells, monocytes, and B cells in dLNs. Similar bimodal dynamics were observed in whole-blood gene signatures in macaques: most of the 17 enriched immune/innate signaling pathways were significantly upregulated at D1 and D7 and downregulated at D3, and clustering analysis revealed stronger similarities between D1 and D7 signatures versus the D3 signature. Serum cytokine responses (CXCL10, IL1Ra, and low-level IFN-α) in macaques were predominantly observed at D1. Altogether, the early immune responses exhibited bimodal kinetics with transient peaks at D1/D2 and D6/D7, mostly with an IFN-associated signature, and these features were remarkably consistent across most analyzed parameters in murine tissues and macaque blood. These compelling observations reveal a novel aspect of the dynamics of innate immunity induced by ChAd155-vectored vaccines, and contribute to ongoing research to better understand how adenovectors can promote vaccine-induced immunity.

Erratum: Author Correction: Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity.

[This corrects the article DOI: 10.1038/s41541-017-0027-3.].

Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity.

Combining immunostimulants in adjuvants can improve the quality of the immune response to vaccines. Here, we report a unique mechanism of molecular and cellular synergy between a TLR4 ligand, 3-O-desacyl-4'-monophosphoryl lipid A (MPL), and a saponin, QS-21, the constituents of the Adjuvant System AS01. AS01 is part of the malaria and herpes zoster vaccine candidates that have demonstrated efficacy in phase III studies. Hours after injection of AS01-adjuvanted vaccine, resident cells, such as NK cells and CD8+ T cells, release IFNγ in the lymph node draining the injection site. This effect results from MPL and QS-21 synergy and is controlled by macrophages, IL-12 and IL-18. Depletion strategies showed that this early IFNγ production was essential for the activation of dendritic cells and the development of Th1 immunity by AS01-adjuvanted vaccine. A similar activation was observed in the lymph node of AS01-injected macaques as well as in the blood of individuals receiving the malaria RTS,S vaccine. This mechanism, previously described for infections, illustrates how adjuvants trigger naturally occurring pathways to improve the efficacy of vaccines.

Enhancement of adaptive immunity by the human vaccine adjuvant AS01 depends on activated dendritic cells.

Adjuvant System AS01 is a liposome-based vaccine adjuvant containing 3-O-desacyl-4'-monophosphoryl lipid A and the saponin QS-21. AS01 has been selected for the clinical development of several candidate vaccines including the RTS,S malaria vaccine and the subunit glycoprotein E varicella zoster vaccine (both currently in phase III). Given the known immunostimulatory properties of MPL and QS-21, the objective of this study was to describe the early immune response parameters after immunization with an AS01-adjuvanted vaccine and to identify relationships with the vaccine-specific adaptive immune response. Cytokine production and innate immune cell recruitment occurred rapidly and transiently at the muscle injection site and draining lymph node postinjection, consistent with the rapid drainage of the vaccine components to the draining lymph node. The induction of Ag-specific Ab and T cell responses was dependent on the Ag being injected at the same time or within 24 h after AS01, suggesting that the early events occurring postinjection were required for these elevated adaptive responses. In the draining lymph node, after 24 h, the numbers of activated and Ag-loaded monocytes and MHCII(high) dendritic cells were higher after the injection of the AS01-adjuvanted vaccine than after Ag alone. However, only MHCII(high) dendritic cells appeared efficient at and necessary for direct Ag presentation to T cells. These data suggest that the ability of AS01 to improve adaptive immune responses, as has been demonstrated in clinical trials, is linked to a transient stimulation of the innate immune system leading to the generation of high number of efficient Ag-presenting dendritic cells.

Development of an AS04-adjuvanted HPV vaccine with the adjuvant system approach.

A novel human papillomavirus (HPV) vaccine has been formulated with virus-like particles of the L1 protein of HPV-16 and HPV-18, and the Adjuvant System 04 (AS04). AS04 is a combination of the toll-like receptor 4 agonist monophosphoryl lipid A (MPL) and aluminum hydroxide. The AS04-adjuvanted HPV vaccine induces a high and sustained immune response against HPV, including high levels of neutralizing antibodies at the cervical mucosa in women aged 15-55 years. Recently, the mechanism of action of AS04 has been evaluated in vitro in human cells and in vivo in mice and the data provide evidence for the molecular and cellular basis of the observed immunogenicity, efficacy, and safety profile of this formulation. In this review, we discuss how the results of GlaxoSmithKline's clinical studies on immunogenicity, protection, and reactogenicity with the AS04-adjuvanted HPV vaccine are supported by the observed mechanism of action for the adjuvant. The adjuvant activity of AS04, as measured by enhanced antibody response to HPV antigens, was found to be strictly dependent on AS04 and the HPV antigens being injected at the same intramuscular site within 24 hours of each other. The addition of MPL to aluminum salt enhances humoral and cell-mediated response by rapidly triggering a local and transient cytokine response that leads to an increased activation of antigen-presenting cells and results in an improved presentation of antigen to CD4+ T cells. The added value of MPL in AS04 for an HPV vaccine was demonstrated in clinical studies by high vaccine-elicited antibody responses and the induction of high levels of memory B cells. The vaccine elicits cross protection against some other oncogenic HPV types (specifically HPV-31, -33, and -45) not contained in the vaccine. The localized and transient nature of the innate immune response supports the acceptable safety profile observed in clinical studies.

Induction of Bordetella pertussis-specific immune memory by DTPa vaccines.

Several vaccines are available against pertussis, differing by the number of Bordetella pertussis antigens that they contain as well as their formulation. The GlaxoSmithKline Biologicals (GSK Bio) tricomponent DTPa vaccine (DTPa3, Infanrix™), and the Sanofi-Pasteur (SP) five-component formulation (DTPa5, Pediacel™) were shown to have comparable short-term efficacy in clinical trials. However, potential differences in long-term protection were recently suggested, which might reflect the elicitation of different specific immune memory by the two vaccines. Therefore, the purpose of the present study was to investigate in mice the immune responses against B. pertussis, and particularly the establishment of specific B cell memory after immunization with DTPa3 and DTPa5 vaccines. Whereas intranasal challenge experiments showed similar protection with both vaccines, DTPa3 induced higher antibody levels to FHA and PRN than DTPa5. Further, the frequency of memory B cells was investigated by B cell ELISPOT. Higher frequencies of PT- and PRN-specific memory B cells were evidenced after vaccination with DTPa3, compared with DTPa5. Although the origin of such difference is unclear, the use of two different adjuvants (aluminum phosphate versus hydroxide) is proposed as a possible explanation. In conclusion, this study proposes that the induction of higher levels of B. pertussis antigen-specific memory B cells with DTPa3 participate to the suggested longer persistence of protection observed with this vaccine, as compared with DTPa5.

Adjuvant System AS03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity.

AS03 is an Adjuvant System (AS) containing α-tocopherol and squalene in an oil-in-water (o/w) emulsion. AS03 has been considered for the development of pandemic and seasonal influenza vaccines. Key features of AS03's mode of action were investigated in vivo in mice and ex vivo in human cells. AS03's adjuvant activity was superior to that of aluminium hydroxide and required the spatio-temporal co-localisation of AS03 with the antigen. This requirement coincided with AS03 triggering a transient production of cytokines at the injection site and in the draining lymph nodes (dLNs). The nature of the cytokines produced was consistent with the enhanced recruitment of granulocytes and of antigen-loaded monocytes in the dLNs. The presence of α-tocopherol in AS03 was required for AS03 to achieve the highest antibody response. The presence of α-tocopherol also modulated the expression of some cytokines, including CCL2, CCL3, IL-6, CSF3 and CXCL1; increased the antigen loading in monocytes; and increased the recruitment of granulocytes in the dLNs. Hence, AS03's promotion of monocytes as the principal antigen-presenting cells, and its effects on granulocytes and cytokines, may all contribute to enhancing the antigen-specific adaptive immune response.

Production of an antigenic peptide by insulin-degrading enzyme.

Most antigenic peptides presented by major histocompatibility complex (MHC) class I molecules are produced by the proteasome. Here we show that a proteasome-independent peptide derived from the human tumor protein MAGE-A3 is produced directly by insulin-degrading enzyme (IDE), a cytosolic metallopeptidase. Cytotoxic T lymphocyte recognition of tumor cells was reduced after metallopeptidase inhibition or IDE silencing. Separate inhibition of the metallopeptidase and the proteasome impaired degradation of MAGE-A3 proteins, and simultaneous inhibition of both further stabilized MAGE-A3 proteins. These results suggest that MAGE-A3 proteins are degraded along two parallel pathways that involve either the proteasome or IDE and produce different sets of antigenic peptides presented by MHC class I molecules.

AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity.

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-kappaB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4(+) T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only.

An effective virus-like particle (VLP) based prophylactic vaccine designed to protect against persistent infection with human papillomavirus (HPV) types 16 and 18 and subsequent lesion development will need to induce a strong humoral and cellular immune response capable of providing long-term protection. Our objective was to evaluate the ability of an HPV16/18 L1 VLP vaccine formulated with the AS04 adjuvant system (3-O-desacyl-4'-monophosphoryl lipid A (MPL) and aluminium salt) to induce an immune response of higher magnitude and persistence compared to a vaccine formulated with aluminium salt only. We demonstrated that MPL adsorbed onto aluminium salt retains its capacity to activate an innate immune response as assessed by the production of TNFalpha by human monocytes (U937). In addition, vaccination of mice, monkeys or human subjects with AS04 formulations induced higher total anti-L1 VLP16 and L1 VLP18 antibody responses (1.6-8.5-fold) than the aluminium salt only formulations. The enhanced antibody response induced by the AS04 vaccine formulation (1.6-4.1-fold) in monkeys and humans was shown to be targeted to functional neutralising L1 VLP16 and L1 VLP18 epitopes as assessed by V5/J4 specific ELISAs or HPV16 and HPV18 pseudo-neutralization assays. The enhanced immune profile observed with the AS04 formulation in terms of both total, V5/J4 specific and neutralizing antibodies was shown to persist for at least 3.5-year post-vaccination in human subjects. Finally, using the newly developed B cell ELISPOT assay we also demonstrated that the AS04 formulation elicited an increased frequency (2.2-5.2-fold) of HPV L1 VLP specific memory B cells when compared with the aluminium salt only formulations. These data strongly support the role of the AS04 adjuvant, which includes the immunostimulant MPL, in triggering a persistent vaccine-induced immune response of high quality.

Processing of tumor-associated antigen by the proteasomes of dendritic cells controls in vivo T-cell responses.

Dendritic cells are unique in their capacity to process antigens and prime naive CD8(+) T cells. Contrary to most cells, which express the standard proteasomes, dendritic cells express immunoproteasomes constitutively. The melanoma-associated protein Melan-A(MART1) contains an HLA-A2-restricted peptide that is poorly processed by melanoma cells expressing immunoproteasomes in vitro. Here, we show that the expression of Melan-A in dendritic cells fails to elicit T-cell responses in vitro and in vivo because it is not processed by the proteasomes of dendritic cells. In contrast, dendritic cells lacking immunoproteasomes induce strong anti-Melan-A T-cell responses in vitro and in vivo. These results suggest that the inefficient processing of self-antigens, such as Melan-A, by the immunoproteasomes of professional antigen-presenting cells prevents the induction of antitumor T-cell responses in vivo.

Destructive cleavage of antigenic peptides either by the immunoproteasome or by the standard proteasome results in differential antigen presentation.

The immunoproteasome (IP) is usually viewed as favoring the production of antigenic peptides presented by MHC class I molecules, mainly because of its higher cleavage activity after hydrophobic residues, referred to as the chymotrypsin-like activity. However, some peptides have been found to be better produced by the standard proteasome. The mechanism of this differential processing has not been described. By studying the processing of three tumor antigenic peptides of clinical interest, we demonstrate that their differential processing mainly results from differences in the efficiency of internal cleavages by the two proteasome types. Peptide gp100(209-217) (ITDQVPSFV) and peptide tyrosinase369-377 (YMDGTMSQV) are destroyed by the IP, which cleaves after an internal hydrophobic residue. Conversely, peptide MAGE-C2(336-344) (ALKDVEERV) is destroyed by the standard proteasome by internal cleavage after an acidic residue, in line with its higher postacidic activity. These results indicate that the IP may destroy some antigenic peptides due to its higher chymotrypsin-like activity, rather than favor their production. They also suggest that the sets of peptides produced by the two proteasome types differ more than expected. Considering that mature dendritic cells mainly contain IPs, our results have implications for the design of immunotherapy strategies.

Two new tumor-specific antigenic peptides encoded by gene MAGE-C2 and presented to cytolytic T lymphocytes by HLA-A2.

We have identified 2 antigens recognized by several melanoma-specific cytolytic T lymphocyte clones isolated from a melanoma patient with a clinical history of tumor regression after immunotherapy. Both antigens are presented by HLA-A2 and encoded by gene MAGE-C2, a cancer-germline gene shown previously to be silent in normal somatic tissues and expressed in 40% of melanomas and in other tumor types. One antigen corresponds to peptide ALKDVEERV(336-344), whereas the other corresponds to peptide LLFGLALIEV(191-200). The CTL clones recognizing these 2 peptides also recognized allogeneic tumor cell lines expressing MAGE-C2 and HLA-A2. These 2 new peptides are the first known MAGE-C antigens and represent promising targets for cancer immunotherapy.

An antigenic peptide produced by peptide splicing in the proteasome.

CD8 T lymphocytes recognize peptides of 8 to 10 amino acids presented by class I molecules of the major histocompatibility complex. Here, CD8 T lymphocytes were found to recognize a nonameric peptide on melanoma cells that comprises two noncontiguous segments of melanocytic glycoprotein gp100(PMEL17). The production of this peptide involves the excision of four amino acids and splicing of the fragments. This process was reproduced in vitro by incubating a precursor peptide of 13 amino acids with highly purified proteasomes. Splicing appears to occur by transpeptidation involving an acyl-enzyme intermediate. Our results reveal an unanticipated aspect of the proteasome function of producing antigenic peptides.

Recombinant adenylate cyclase toxin of Bordetella pertussis induces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes.

The adenylate cyclase (CyaA) of Bordetella pertussis is able to deliver CD8(+) T cell epitopes into the cytosol of CD11b(+) dendritic cells (DC) following its specific interaction with the alpha(M)beta(2) integrin (CD11b/CD18). This delivery results in intracellular processing and presentation by MHC class I molecules of the CD8(+) T cell epitopes inserted into CyaA. Indeed, we previously showed that CyaA toxins carrying a single cytotoxic T lymphocyte (CTL) epitope can induce efficient protective and therapeutic antitumor immunity in mice. With a view to elaborating cancer immunotherapy in humans using CyaA, we constructed two recombinant CyaA carrying HLA*0201-restricted melanoma epitopes. Here we show that these recombinant CyaA induce strong anti-melanoma CTL responses in HLA*0201 transgenic mice, even after a single i.v. immunization without adjuvant. These responses are long lasting, since they were also detected 5 months after the last injection. Finally, human DC treated with the recombinant CyaA were shown to process and present efficiently the melanoma epitopes to human CTL clones. Altogether, our results demonstrate that tumoral epitopes inserted into CyaA are efficiently processed and presented in association with human MHC molecules. These observations suggest that CyaA is capable of activating antitumoral CTL in humans and highlight the potential of CyaA for use in cancer immunotherapy.

The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in the cytosol is mediated by two peptidases.

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.

The production of a new MAGE-3 peptide presented to cytolytic T lymphocytes by HLA-B40 requires the immunoproteasome.

By stimulating human CD8(+) T lymphocytes with autologous dendritic cells infected with an adenovirus encoding MAGE-3, we obtained a cytotoxic T lymphocyte (CTL) clone that recognized a new MAGE-3 antigenic peptide, AELVHFLLL, which is presented by HLA-B40. This peptide is also encoded by MAGE-12. The CTL clone recognized MAGE-3--expressing tumor cells only when they were first treated with IFN-gamma. Since this treatment is known to induce the exchange of the three catalytic subunits of the proteasome to form the immunoproteasome, this result suggested that the processing of this MAGE-3 peptide required the immunoproteasome. Transfection experiments showed that the substitution of beta5i (LMP7) for beta5 is necessary and sufficient for producing the peptide, whereas a mutated form of beta5i (LMP7) lacking the catalytically active site was ineffective. Mass spectrometric analyses of in vitro digestions of a long precursor peptide with either proteasome type showed that the immunoproteasome produced the antigenic peptide more efficiently, whereas the standard proteasome more efficiently introduced cleavages destroying the antigenic peptide. This is the first example of a tumor-specific antigen exclusively presented by tumor cells expressing the immunoproteasome.