CD161(int)CD8+ T cells: a novel population of highly functional, memory CD8+ T cells enriched within the gut.

The C-type lectin-like receptor CD161 is expressed by lymphocytes found in human gut and liver, as well as blood, especially natural killer (NK) cells, T helper 17 (Th17) cells, and a population of unconventional T cells known as mucosal-associated invariant T (MAIT) cells. The association of high CD161 expression with innate T-cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including antiviral populations that display a memory phenotype. These memory CD161(int)CD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterized by enhanced polyfunctionality, increased levels of cytotoxic mediators, and high expression of the transcription factors T-bet and eomesodermin (EOMES). Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T-cell populations in humans. As induction of such responses represents a major aim of T-cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

Immune complexed (IC) hepatitis C virus (HCV) in chronically and acutely HCV-infected patients.

In infected individuals, hepatitis C virus (HCV) exists in various forms of circulating particles which role in virus persistence and in HCV resistance to IFN therapy is still debated. Here, the proportion of HCV bound to immunoglobulin was determined in plasma of 107 chronically infected patients harbouring different HCV genotypes and, for comparison, of six patients with acute HCV infection. The results showed that, in spite of wide individual variability, chronically HCV-infected patients exhibited an extremely high proportion of immune complexed (IC) virus regardless of plasma HCV load and infecting genotype. Moreover, no significant association was found between baseline proportion of IC HCV and response to IFN treatment. Plasma samples collected within 2 weeks of treatment from 20 patients revealed a significant decline of mean IC HCV values relative to baseline that clearly paralleled the decay of total HCV load. In acutely infected patients, circulating HCV was not IC or IC at very low levels only in patients developing chronic HCV infection. Collectively, these findings strengthen the possibility that IC virus could play a critical role in the pathogenesis of HCV infection.

Identification of four novel MHC-C alleles in chimpanzees.

Sequence-based typing strategy is used to identify polymorphism of Patr-C. Four new Patr-C alleles are described.

Hepatitis C virus carriers with persistently normal ALT levels: biological peculiarities and update of the natural history of liver disease at 10 years.

Some chronic hepatitis C (CHC) patients exhibit persistently normal alanine aminotransferase (ALT) levels (PNAL). Patients with PNAL experience significantly milder disease. In order to understand the differences between CHC patients with elevated ALT levels compared with those with PNAL better, we compared epidemiological, immunological and histological findings, in particular, the value of proliferating hepatocyte activity (PCNA) between the two groups of patients. We studied 40 chronic hepatitis C virus (HCV) carriers with increased ALT who underwent liver biopsy for histological diagnosis and determination of clinical prognosis, and 24 PNAL patients under follow-up for 10 years. Immunological response to different HCV genomic epitopes was tested in both the control group and in PNAL subjects. PCNA values from liver specimens of all patients as well as liver biopsies of PNAL patients at time points 0 and 5 years were calculated according to Hall et al.Age, sex and body mass index (BMI) were not significantly different between the two groups. The median liver histology stage was significantly higher in HCV carriers vs the PNAL group (2.5, range = 2-6 vs 1.5, range = 1-2; P < 0.01). Among PNAL patients, histological stage was not statistically different at the three time points considered. Interferon (IFN)-gamma production was comparable in the two groups. PCNA was significantly higher in the group with elevated ALT levels vs the PNAL group (8%, range = 4-15%vs 5% range = 3-8%; P < 0.05) and no statistically significant differences were found in PNAL patients at time points 0, 5 and 10 years. This study confirms that progression to cirrhosis is slow or absent in PNAL patients after 10 years of follow-up. Accordingly, the hepatic proliferative activity index is low and seems to be stable over time.

Efficient immunization of rhesus macaques with an HCV candidate vaccine by heterologous priming-boosting with novel adenoviral vectors based on different serotypes.

Efficient vaccination against viral agents requires a strong T-cell-mediated immune response to clear viral-infected cells. Optimal vaccination can be achieved by administration of recombinant viral vectors encoding phatogen antigens. Adenoviral vectors have attracted considerable attention as potential viral vectors for genetic vaccination owing to their favorable safety profile and potent transduction efficiency following intramuscular injection. However, the neutralizing antibody response against adenoviral capsid proteins following adenoviral vectors injection limits the success of vaccination protocols based on multiple administrations of the same adenoviral serotype. In this work, we describe efficient immunization of rhesus macaques, the preferred model for preclinical assessment, with an HCV candidate vaccine by heterologous priming-boosting with adenoviral vectors based on different serotypes. The induced responses are broad and show significant cross-strain reactivity. Boosting can be delayed for over 2 years after priming, indicating that there is long-term maintenance of resting memory cells.

Early impairment of hepatitis C virus specific T cell proliferation during acute infection leads to failure of viral clearance.

BACKGROUND AND AIMS: Cellular mediated immunity (CMI) is thought to play a key role in resolution of primary hepatitis C virus (HCV) infection. However, CD4+ and CD8+ T cell responses are also generated during acute infection in individuals who become chronic, suggesting that they developed a defective CMI. The aim of this study was to verify if and when such immune dysfunction is established by measuring the breadth, magnitude, function, and duration of CMI in a large cohort of subjects during the natural course of acute HCV infection. METHODS: CMI was comprehensively studied by prospective sampling of 31 HCV acutely infected subjects enrolled at the onset of infection and followed for a median period of one year. RESULTS: Our results indicated that while at the onset of acute HCV infection a measurable CMI with effector function was detected in the majority of subjects, after approximately six months less than 10% of chronically infected individuals displayed significant CMI compared with 70% of subjects who cleared the virus. We showed that progressive disappearance of HCV specific T cells from the peripheral blood of chronic patients was due to an impaired ability to proliferate that could be rescued in vitro by concomitant exposure to interleukin 2 and the antigen. CONCLUSION: Our data provide evidence of strong and multispecific T cell responses with a sustained ability to proliferate in response to antigen stimulation as reliable pharmacodynamic measures of a protective CMI during acute infection, and suggest that early impairment of proliferation may contribute to loss of T cell response and chronic HCV persistence.

Multispecific T cell response and negative HCV RNA tests during acute HCV infection are early prognostic factors of spontaneous clearance.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection results in a high frequency of chronic disease. The aim of this study was to identify early prognostic markers of disease resolution by performing a comprehensive analysis of viral and host factors during the natural course of acute HCV infection. METHODS: The clinical course of acute hepatitis C was determined in 34 consecutive patients. Epidemiological and virological parameters, as well as cell mediated immunity (CMI) and distribution of human leukocyte antigens (HLA) alleles were analysed. RESULTS: Ten out of 34 patients experienced self-limiting infection, with most resolving patients showing fast kinetics of viral clearance: at least one negative HCV RNA test during this phase predicted a favourable outcome. Among other clinical epidemiological parameters measured, the self-limiting course was significantly associated with higher median peak bilirubin levels at the onset of disease, and with the female sex, but only the latter parameter was independently associated after multivariate analysis. No significant differences between self-limiting or chronic course were observed for the distribution of DRB1 and DQB1 alleles. HCV specific T cell response was more frequently detected during acute HCV infection, than in patients with chronic HCV disease. A significantly broader T cell response was found in patients with self-limiting infection than in those with chronic evolving acute hepatitis C. CONCLUSION: The results suggest that host related factors, in particular sex and CMI, play a crucial role in the spontaneous clearance of this virus. Most importantly, a negative HCV RNA test and broad CMI within the first month after onset of the symptoms represent very efficacious predictors of viral clearance and could thus be used as criteria in selecting candidates for early antiviral treatment.

Mimotopes of the hyper variable region 1 of the hepatitis C virus induce cross-reactive antibodies directed against discontinuous epitopes.

Hepatitis C virus (HCV) is a major cause worldwide of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, and the development of an effective vaccine represents a high priority goal. The hyper variable region 1 (HVR1) of the second envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. To be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1, selected from a specialized phage library using HCV patients' sera. Some of the cross-reacting anti-mimotope antibodies elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.

Induction of cross-reactive humoral immune response by immunization with mimotopes of the hypervariable region 1 of the hepatitis C virus.

Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, worldwide, and the development of an effective vaccine represents a high priority goal. The Hyper Variable Region 1 (HVR1) of the second Envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. Thus, to be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1. selected from a specialized phage library using HCV patients' sera. At least some of the cross-reacting anti-mimotope antibodies, elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.

Enhancing B- and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscular electrical gene transfer.

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8(+) T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

Biochemical and immunologic properties of the nonstructural proteins of the hepatitis C virus: implications for development of antiviral agents and vaccines.

Infection with the hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. The viral genome, a positive-sense, single-stranded, 9.6-kb long RNA molecule, is translated into a single polyprotein of about 3,000 amino acids. The viral polyprotein is proteoytically processed to yield all the mature viral gene products. The genomic order of HCV has been determined to be C-->E1-->E2-->p7-->NS2-->NS3-->NS4A-->NS4B-->NS5A++ +-->NS5B. C, E1, and E2 are the virion structural proteins. Whereas the function of p7 is currently unknown, NS2 to NS5B are thought to be the nonstructural proteins. Generation of the mature nonstructural proteins relies on the activity of viral proteinases. Cleavage at the NS2-NS3 junction is accomplished by a metal-dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining downstream cleavages are effected by a serine proteinase contained also within the N-terminal region of NS3. NS3, in addition, contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that acts as a cofactor of the proteinase. Although no function has yet been attributed to NS4B, NS5A has been recently suggested to be involved in mediating the resistance of the HCV to the action of interferon. Finally, the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase. This article reviews the current understanding of the structure and the function of the various HCV nonstructural proteins with particular emphasis on their potential as targets for the development of novel antiviral agents and vaccines.

Induction of anti-carbohydrate antibodies by phage library-selected peptide mimics.

One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T-independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage-displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O-antigen (O-Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage-displayed nonapeptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme-linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3-specific) and p115 (interacting with both mIgA) were able to induce anti-O-Ag antibodies. The immune response was specific for the O-Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPLGALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti-polysaccharide vaccines.

A conformationally homogeneous combinatorial peptide library.

In search for a rational way to convert the information encoded in peptide structures into peptidomimetics, major progress could be made by coupling the power of selection methods, now enormously increased in number as a result of the development of combinatorial peptide libraries, with the rational design of structure-inducing templates for the selectable sequences. The availability of libraries of peptides with predetermined structure would enable selection-driven peptidomimetic design, whereby a conformational model for the peptide pharmacophore would be directly derived from the screening, allowing the design of a suitable non-peptidic scaffold to replace the peptide backbone. We describe here the first example of a conformationally homogeneous combinatorial peptide library, which yields ligands with the expected structure upon selection. The library was built by randomising five positions in the alpha-helical portion of a 26 amino acid Cys2His2 consensus "zinc-finger" motif. Since in zinc-fingers metal coordination and folding are coupled, in our library metal-dependent binding represents a built-in control against the selection of structurally undefined sequences. The alpha-helical library was produced as both fusion with the pVIII protein of filamentous phage and soluble peptides by chemical synthesis, the latter enabling the expansion of the selectable repertoire by the inclusion of non-coded amino acids. The two libraries were independently screened with the same receptor (a monoclonal IgA reactive against the lipopolysaccharide of the human pathogen Shigella flexneri), yielding a very similar consensus. In particular, the peptides defined by both methods showed very strong, zinc-dependent binding to the IgA. The geometrical arrangement of the side-chains of the selected peptide pharmacophore was shown by circular dichroism, Co(II)-complex absorption and high-resolution NMR to be structurally invariant with respect to the parent zinc-finger.

Monoclonal antibodies that recognise filamentous phage: tools for phage display technology.

We generated six hybridoma cell lines that secrete monoclonal antibodies (mAb) which specifically bind filamentous phage coat proteins. Two of these mAb recognise epitopes that include the N terminus of the coat protein III (pIII), while two others are specific for the N terminus of the major coat protein VIII (pVIII). These mAb are valuable tools to study phage assembly and structure. Furthermore, we describe two examples of how these mAb can be exploited in the construction and screening of peptide libraries displayed by the filamentous phase major coat protein. We have used one of these mAb to develop a sensitive ELISA with crude phage supernatants. This assay allows rapid screening of large numbers of clones from random peptide phage libraries. Some of the anti-phage mAb described here can interfere with wild-type phage propagation, while phage carrying modifications in their coat proteins are insensitive to growth inhibition. We have exploited this observation as a tool to favour the growth of phage displaying peptides fused to pVIII, with respect to vector phage.

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease-specific epitopes from phage-displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease-specific phagotopes can be identified out of this pool through an 'antigen independent' procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease-related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

Mimicking of discontinuous epitopes by phage-displayed peptides, II. Selection of clones recognized by a protective monoclonal antibody against the Bordetella pertussis toxin from phage peptide libraries.

We have screened phage peptide libraries to establish if clones binding to a monoclonal antibody (mAb), specific for a discontinuous epitope, could be isolated and if the selected phage particles would be able to elicit an in vivo immuno-response against the original antigen. Two phage peptide libraries, consisting of 9 random amino acids inserted in the major coat protein (pVIII), were independently screened with a mAb which is capable of neutralizing the Bordetella pertussis toxin (PTX) in in vitro and in vivo assays. The epitope of PTX recognized by this and other protective mAb has been shown to be discontinuous. Six different positive phage clones were selected; their binding to the mAb could be competed for by PTX, showing that these clones bind to the antigen-binding site of the mAb. Three of the clones were used (alone or as a mixture) to immunize BALB/c mice. The sera showed a good immunoresponse both against the phage bearing the epitopes and against synthetic multiple-antigen peptides of the same sequence. The immune sera, however, showed no detectable signal against PTX and no capacity to neutralize the CHO-cell-clustering activity of the toxin. The results show that the selected recombinant phage are capable of mimicking the discontinuous epitope as far as binding to the corresponding mAb, but they are unable to elicit a detectable production of antibodies specific for the original antigen.