OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its close homologue, OTUB2, suggesting variations in regulatory mechanisms and substrate specificity. Biochemical analysis reveals that OTUB1 has a preference for cleaving Lys(48)-linked polyubiquitin chains over Lys(63)-linked polyubiquitin chains, and it is capable of cleaving NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), but not SUMO (small ubiquitin-related modifier) 1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates. A functional comparison of OTUB1 and OTUB2 indicated a differential reactivity towards ubiquitin-based active-site probes carrying a vinyl methyl ester, a 2-chloroethyl or a 2-bromoethyl group at the C-terminus. Mutational analysis suggested that a narrow P1' site, as observed in OTUB1, correlates with its ability to preferentially cleave Lys(48)-linked ubiquitin chains. Analysis of cellular interaction partners of OTUB1 by co-immunoprecipitation and MS/MS (tandem mass spectrometry) experiments demonstrated that FUS [fusion involved in t(12;6) in malignant liposarcoma; also known as TLS (translocation in liposarcoma) or CHOP (CCAAT/enhancer-binding protein homologous protein)] and RACK1 [receptor for activated kinase 1; also known as GNB2L1 (guanine-nucleotide-binding protein beta polypeptide 2-like 1)] are part of OTUB1-containing complexes, pointing towards a molecular function of this deubiquitinating enzyme in RNA processing and cell adhesion/morphology.

All thymically selected T cells are inherently cross-reactive, yet many data indicate a fine specificity in antigen recognition, which enables virus escape from immune control by mutation in infections such as the human immunodeficiency virus (HIV). To address this paradox, we analyzed the fine specificity of T cells recognizing a human histocompatibility leukocyte antigen (HLA)-A2-restricted, strongly immunodominant, HIV gag epitope (SLFNTVATL). The majority of 171 variant peptides tested bound HLA-A2, but only one third were recognized. Surprisingly, one recognized variant (SLYNTVATL) showed marked differences in structure when bound to HLA-A2. T cell receptor (TCR) recognition of variants of these two peptides implied that they adopted the same conformation in the TCR-peptide-major histocompatibility complex (MHC) complex. However, the on-rate kinetics of TCR binding were identical, implying that conformational changes at the TCR-peptide-MHC binding interface occur after an initial permissive antigen contact. These findings have implications for the rational design of vaccines targeting viruses with unstable genomes.

We have solved the crystal structures of three HLA‐B*2705–peptide complexes with the immunodominant viral peptides: EBV EBNA3C 258–266 (RRIYDLIEL), influenza (flu) nucleoprotein NP383–391 (SRYWAIRTR), and HIV gag 264–273 (KRWIILGLNK). Long‐term non‐progression during HIV infection has been associated with presentation by HLA‐B*2705, and T cell recognition, of the highly immunodominant KRWIILGLNK peptide. The tight hydrogen‐bonding network observed between the HLA‐B*2705 B‐pocket and the peptide P2 arginine guanadinium anchor explains why mutation of this residue during HIV infection results in loss of peptide binding, immune escape and progression to AIDS. Prominent, solvent‐exposed structures within these peptides may participate in generating T cell responses to these immunodominant epitopes. In the HLA‐B*2705 complex with flu NP383–391, the amino acid side chains of residues 4, 7 and 8 are solvent‐exposed whilst in the HIV decamer, the main‐chain bulges into the solvent around P7. Thus, HLA‐B*2705 presents viral peptides in a range of conformations. Tetrameric complexes of HLA‐B*2705 with the HIV and flu but not EBV peptides bound strongly to the killer‐Ig‐like receptor (KIR)3DL1. Substitution of EBV P8 glutamate to threonine allowed recognition by KIR3DL1. In the HLA‐B*2705–EBV structure the P8 glutamate side chain is solvent‐exposed and may inhibit KIR3DL1 binding through electrostatic forces.See accompanying Commentary: http://dx.doi.org/10.1002/eji.200425875

HLA-B27 can form beta-2 microglobulin (b2m)-associated heterotrimers (HLA-B27) and b2m-free homodimers (B27 2 ). Here, we study the role of complexed peptide in the interaction of these forms of B27 with the killer cell immunoglobulin (Ig)-like receptors KIR3DL1 and KIR3DL2 and with Ig-like transcripts LILRB1 and LILRB2. HLA-B27 tetramers complexed with three of five different naturally processed self peptides and three of seven pathogen-derived epitopes bound to KIR3DL1-expressing transfectants and NK cells. Heterotrimeric complexes containing peptides with charged amino acids at position 8 did not bind to KIR3DL1; however, studies with analogue peptides demonstrated that these are not the only peptide residues involved in binding. KIR3DL1 ligation by HLA-B27 inhibited NK cell IFN-c production in a peptide-dependent fashion. B27 but not HLA-A2, B7 or B57 heavy chains formed homodimers in the presence of peptide epitopes. B27 2 bound to KIR3DL1, KIR3DL2 and LILRB2 but not LILRB1. KIR3DL2 ligation by B27 2 inhibited NK and T cell IFN-c production. By contrast with HLA heterotrimers, B27 2 binding to KIR did not depend on the sequence of the bound peptide. Differences in KIR binding to classical HLA and B27 2 could be involved in the pathogenesis of spondyloarthritis.

Affordable therapeutic strategies that induce sustained control of human immunodeficiency virus type 1 (HIV-1) replication and are tailored to the developing world are urgently needed. Since CD8 ؉ and CD4 ؉ T cells are crucial to HIV-1 control, stimulation of potent cellular responses by therapeutic vaccination might be exploited to reduce antiretroviral drug exposure. However, therapeutic vaccines tested to date have shown modest immunogenicity. In this study, we performed a comprehensive analysis of the changes in virus-specific CD8 ؉ and CD4 ؉ T-cell responses occurring after vaccination of 16 HIV-1-infected individuals with a recombinant modified vaccinia virus Ankara-vectored vaccine expressing the consensus HIV-1 clade A Gag p24/p17 sequences and multiple CD8 ؉ T-cell epitopes during highly active antiretroviral therapy. We observed significant amplification and broadening of CD8 ؉ and CD4 ؉ gamma interferon responses to vaccine-derived epitopes in the vaccinees, without rebound viremia, but not in two unvaccinated controls followed simultaneously. Vaccine-driven CD8 ؉ T-cell expansions were also detected by tetramer reactivity, predominantly in the CD45RA ؊ CCR7 ؉ or CD45RA ؊ CCR7 ؊ compartments, and persisted for at least 1 year. Expansion was associated with a marked but transient up-regulation of CD38 and perforin within days of vaccination. Gag-specific CD8؉ and CD4 ؉ T-cell proliferation also increased postvaccination. These data suggest that immunization with MVA

The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, β-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5–7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha–1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

To better understand the mechanisms of intracellular trafficking and presentation of exogenous peptides by antigen-presenting cells (APC), we compared the handling of overlapping 24-mer peptides from HIV Nef either mixed or covalently linked in tandem in one protein. Once internalized, peptides trafficked not only to endosomes but also to cytosol, and activated CD8 ؉ and CD4؉ T cells. In contrast, whole protein was found to traffic only to the endosomal compartments, and primarily activated CD4 ؉ T cells. Finally, with adjuvant, overlapping peptides were capable of protecting against lethal viral challenge, whereas the intact protein was less protective. These data suggest that overlapping long peptides are cross-presented through more varied intracellular routes and are more efficient in priming protective immunity than the whole protein.Despite much progress in vaccine development, there are still several challenges for design of subunit vaccines. Against intracellular pathogens, it is especially important to immunize protective CD4 ϩ and CD8 ϩ T cell responses. The former recognize epitopes presented by major histocompatibility complex (MHC) 3 class II molecules that are normally loaded mainly with peptides derived from exogenous antigens. Natural processing and loading are catalyzed by HLA-or H2-DM in acidic lysosomes (1).By contrast, CD8 ϩ T cell responses typically depend on epitope loading via the class I pathway. This "endogenous" route starts from the cytosol of any cells naturally infected by the relevant virus, which may not include professional antigen presenting cells (pAPCs). Once processed by cytosolic proteases, viral peptide fragments are transported into the endoplasmic reticulum, and loaded onto MHC class I molecules, which then traffic to the surface where they can present to CD8 ϩ T cells (2, 3). For exogenous antigens, "cross-presentation" by pAPCs is essential for priming CD8 ϩ as well as CD4 ϩ T cells.Although they must be crucial for most CD8 ϩ T cell vaccination strategies, the mechanisms underlying cross-presentation are not understood. First, internalized antigens are known to enter the cytoplasm, although the mechanisms remain unknown. Once in the cytoplasm, they can follow the conventional MHC class I pathway, i.e. processing by cytosolic proteasomes or proteases, transport into the endoplasmic reticulum, loading onto MHC class I molecules, and then trafficking to the surface for recognition by T cells (2, 3). Second, antigens internalized into endocytic compartments could be degraded by the local proteases into peptides and then loaded onto MHC class I molecules in the endocytic compartments that are recycled to and from the cell surface (4). Third, endosomes may fuse with the endoplasmic reticulum, allowing direct access of such peptides for loading onto nascent MHC class I molecules there before presentation at the cell surface (5); however, this theory remains controversial (6, 7).It has been hypothesized that endocytic and cytosolic proteases process antigens differently. If ...

Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.