Kati di Gleria

T Cell Cross-Reactivity and Conformational Changes during TCR Engagement

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.

Interaction of HLA-B27 homodimers with KIR3DL1 and KIR3DL2, unlike HLA-B27 heterotrimers, is independent of the sequence of bound peptide.

HLA‐B27 can form beta‐2 microglobulin (β2m)‐associated heterotrimers (HLA‐B27) and β2m‐free homodimers (B272). 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‐γ production in a peptide‐dependent fashion. B27 but not HLA‐A2, B7 or B57 heavy chains formed homodimers in the presence of peptide epitopes. B272 bound to KIR3DL1, KIR3DL2 and LILRB2 but not LILRB1. KIR3DL2 ligation by B272 inhibited NK and T cell IFN‐γ production. By contrast with HLA heterotrimers, B272 binding to KIR did not depend on the sequence of the bound peptide. Differences in KIR binding to classical HLA and B272 could be involved in the pathogenesis of spondyloarthritis.

Expansion and Diversification of Virus-Specific T Cells following Immunization of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals with a Recombinant Modified Vaccinia Virus Ankara/HIV-1 Gag Vaccine

ABSTRACT 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.HIVA is a feasible strategy to enhance potentially protective T-cell responses in individuals with chronic HIV-1 infection.

An Early HIV Mutation within an HLA-B*57-Restricted T Cell Epitope Abrogates Binding to the Killer Inhibitory Receptor 3DL1

ABSTRACT 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.

Immunisation with recombinant modified vaccinia virus Ankara expressing HIV-1 gag in HIV-1-infected subjects stimulates broad functional CD4+ T cell responses.

Virus‐specific CD4+ T cells with IL‐2‐secreting and/or proliferative capacity are detected readily in HIV‐1‐infected long‐term nonprogressors and rarely in persons with untreated progressive infection. The contribution of these cells to viraemia control is uncertain, but this question might be addressed in clinical therapeutic vaccination studies. However, the quality of T helper responses induced by currently available HIV‐1 vaccine candidates has not been explored in depth. We determined the effect of vaccination with modified vaccinia virus Ankara (MVA) expressing HIV‐1 gag p24/p17 (MVA.HIVA) on HIV‐1‐specific CD4+ T cell responses in 16 chronically infected, highly active antiretroviral therapy (HAART)‐treated subjects using CD8‐depleted IFN‐γ ELISPOT assays, intracellular cytokine staining assays for IL‐2 and IFN‐γ, and a CFSE‐based proliferation assay. Gag‐specific CD4+ T cell responses were significantly increased in magnitude and breadth after vaccination and targeted both known and new epitopes, several of which were also recognised by healthy HIV‐uninfected volunteers immunised with the same vaccines. The frequencies of CD4+ T cells expressing IL‐2 or IFN‐γ, alone or simultaneously, were also augmented. These findings indicate that functional virus‐specific T helper cells can be boosted by vaccination in chronic HIV‐1 infection. Further evaluation of their role in viraemia control is warranted.

Induction of long-lasting multi-specific CD8+ T cells by a four- component DNA-MVA/HIVA/RENTA candidate HIV-1 vaccine in rhesus macaques

As a part of a long‐term effort to develop vaccine against HIV‐1 clade A inducing protective T cell responses in humans, we run mutually complementing studies in humans and non‐human primates (NHP) with the aim to maximize vaccine immunogenicity. The candidate vaccine under development has four components, pTHr.HIVA and pTH.RENTA DNA, and modified vaccinia virus Ankara (MVA).HIVA and MVA.RENTA, delivered in a heterologous DNA prime‐MVA boost regimen. While the HIVA (Gag/epitopes) components have been tested in NHP and over 300 human subjects, we plan to test in humans the RENTA (reverse transcriptase, gp41, Nef, Tat) vaccines designed to broaden HIVA‐induced responses in year 2007. Here, we investigated the four‐component vaccine long‐term immunogenicity in Mamu‐A*01‐positive rhesus macaques and demonstrated that the vaccine‐induced T cells were multi‐specific, multi‐functional, readily proliferated to recall peptides and were circulating in the peripheral blood of vaccine recipients over 1 year after vaccine administration. The consensus clade A‐elicited T cells recognized 50% of tested epitope variants from other HIV‐1 clades. Thus, the DNA‐MVA/HIVA‐RENTA vaccine induced memory T cells of desirable characteristics and similarities to those induced in humans by HIVA vaccines alone; however, single‐clade vaccines may not elicit sufficiently cross‐reactive responses.

Homogeneous amperometric ligand-binding assay amplified by a proteolytic enzyme cascade

A homogeneous electrochemically-linked ligand binding assay with enhanced sensitivity is described. A proteolytic enzyme cascade was used to amplify the signal. The avidin-biotin interaction serves to model an immunochemical reaction. Biotinylated trypsin (BT) is the trigger enzyme of the cascade, which is inhibited in the presence of avidin; this inhibition is reversed by the addition of free biotin. The liberated BT activates catalytically the second enzyme, chymotrypsin, which acts upon its immobilised electroactive substrate. The released hydrolysis product, ferrocenoyltyrosine (FcT), is then coupled to the turnover of glucose oxidase (GOx), providing a further level of amplification. The catalytic current produced is proportional to the concentration of biotin in the sample and is linear in the concentration range 0.4–5 μM.

Interleukin-4 promotes human CD8+ T cell expression of CCR7

Despite strong evidence supporting a pathway of human T cell differentiation characterized by changes in the expression of CCR7, CD28, CD27 and CD62L, few studies have addressed the mechanisms of pathway regulation. Cutaneous lymphocyte‐associated antigen (CLA)‐positive skin‐homing CD8+ T cells expressed significantly elevated levels of activation markers compared with CLA− CD8+ T cells in individuals (n = 27) with cutaneous atopic disease. Despite such an activated phenotype, CLA+ T cells expressed significantly higher levels of CCR7 than a CLA− T cell subset. Interleukin (IL)‐4 was found to dramatically promote CCR7 expression by antigen‐specific CD8+ cells. Furthermore, skin‐homing CD8+ T cells from individuals with severe disease produced significantly less IL‐10 than those derived from mildly affected atopic subjects. Thus in a T‐helper 2 dominated disease, tissue‐specific CD8+ T cells show altered CCR7 expression and cytokine production, which may contribute to continued lymph node homing, antigen presentation and disease. IL‐4 promotes expression of CCR7, a marker linked to existing models of CD8+ T cell differentiation.

Covalent linkage of glucose oxidase to modified basal plane pyrolytic graphite electrodes and the use in the ferrocene-mediated amperometric measurement of glucose

A simple, fast and effective method for the immobilization of glucose oxidase onto a modified basal plane pyrolytic graphite electrode is described. To enhance the rate of electron exchange with the electrode, a ferrocene derivative was used as a mediator. The stability of the enzyme electrode was evaluated using direct current cyclic voltammetry.