HCV E1E2-MF59 vaccine in chronic hepatitis C patients treated with PEG-IFNα2a and Ribavirin: a randomized controlled trial.

Hepatitis C virus (HCV) vaccines may be able to increase viral clearance in combination with antiviral therapy. We analysed viral dynamics and HCV-specific immune response during retreatment for experienced patients in a phase Ib study with E1E2MF59 vaccine. Seventy-eight genotype 1a/1b patients [relapsers (30), partial responders (16) and nonresponders (32) to interferon-(IFN)/ribavirin-(RBV)] were randomly assigned to vaccine (V:23), Peg-IFNα2a-180-ug/qw and ribavirin 1000-1200-mg/qd for 48 weeks (P/R:25), or their combination (P/R + V:30). Vaccine (100 µg/0.5 mL) was administered intramuscularly at week 0-4-8-12-24-28-32-36. Neutralizing of binding (NOB) antibodies and lymphocyte proliferation assay (LPA) for E1E2-specific-CD4 + T cells were performed at week 0-12-16-48. Viral kinetics were analysed up to week 16. The vaccine was safe, and a sustained virological response (SVR) was achieved in 4 P/R + V and 2 P/R patients. Higher SVR rates were observed in prior relapsers (P/R + V = 27.3%; P/R = 12.5%). Higher NOB titres and LPA indexes were found at week 12 and 16 in P/R + V as compared to P/R patients (P = 0.023 and 0.025, P = 0.019 and <0.001, respectively). Among the 22 patients with the strongest direct antiviral effects of IFN (ε ≥ 0.800), those treated with P/R + V (10) reached lower HCV-RNA levels (P = 0.026) at week 16. HCV E1E2MF59 vaccine in combination with Peg-IFNα2a + RBV was safe and elicited E1E2 neutralizing antibodies and specific CD4 + T cell proliferation. Upon early response to IFN, vaccinations were associated with an enhanced second phase viral load decline. These results prompt phase II trials in combination with new antiviral therapies.

Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4+ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C.

The adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4+ T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCR) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCR identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCR transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein NS4 is highly immunogenic for intrahepatic CD4+ T cells primed by HCV in vivo, and that there can be compartmentalization of some NS4-specific CD4+ T cells to the liver of patients with chronic hepatitis C.

Anisotropic mechanical response of layered disordered fibrous materials.

Mechanically bonded fabrics account for a significant portion of nonwoven products, and serve many niche areas of nonwoven manufacturing. Such fabrics are characterized by layers of disordered fibrous webs, but we lack an understanding of how such microstructures determine bulk material response. Here we numerically determine the linear shear response of needle-punched fabrics modeled as cross-linked sheets of two-dimensional (2D) Mikado networks. We systematically vary the intra-sheet fiber density, inter-sheet separation distance, and direction of shear, and quantify the macroscopic shear modulus alongside the degree of affinity and energy partition. For shear parallel to the sheets, the response is dominated by intrasheet fibers and follows known trends for 2D Mikado networks. By contrast, shears perpendicular to the sheets induce a softer response dominated by either intrasheet or intersheet fibers depending on a quadratic relation between sheet separation and fiber density. These basic trends are reproduced and elucidated by a simple scaling argument that we provide. We discuss the implications of our findings in the context of real nonwoven fabrics.

Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.

A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.

Binding of hepatitis C virus to CD81.

Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Quantitative analysis of the peripheral blood cytotoxic T lymphocyte response in patients with chronic hepatitis C virus infection.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are present in the peripheral blood and liver of chronically infected patients. The current study was performed to study the relationship between the strength of the CTL response, liver disease severity, and viral load. The results may be summarized as follows: first, using CTL precursor frequency (CTLpf) analysis to quantitate the peripheral blood CTL response, chronically infected patients were less strongly sensitized to a panel of well-defined HCV epitopes than they were to an epitope within the influenza matrix protein. Second, HCV-specific CTLpf did not correlate with disease activity or viral load in the majority of patients on a cross-sectional basis, although it did increase in three patients concomitant with sharp increases in liver disease. Finally, interferon therapy did not enhance the CTLpf against the HCV epitopes studied in these patients, indicating that its antiviral effect is independent of the CTL response. Since the HCV-specific CTLpf in the blood is actually quite low, the CTL may contribute to ongoing liver disease in these patients while being quantitatively inadequate to destroy all of the infected hepatocytes, thereby facilitating HCV persistence and contributing to chronic liver disease.

Cytotoxic T lymphocyte response to hepatitis C virus-derived peptides containing the HLA A2.1 binding motif.

The HLA class I-restricted cytotoxic T lymphocyte (CTL) response is a major defense mechanism in viral infections. It has been suggested that the CTL response may contribute to viral clearance and liver cell injury during hepatitis C virus (HCV) infection. To test this hypothesis requires an understanding of the characteristics of HCV-specific cytotoxic effector cells and identification of the target antigens to which they respond. To begin this process we stimulated peripheral blood mononuclear cells (PBMC) from a group of HLA-A2 positive patients with chronic hepatitis C with a panel of 130 HCV-derived peptides containing the HLA-A2 binding motif. Effector cells were tested for their capacity to lyse HLA-A2-matched target cells that were either sensitized with peptide or infected with a vaccinia virus construct containing HCV sequences. Using this approach we have identified nine immunogenic peptides in HCV, three of which are derived from the putative core protein, three from the nonstructural (NS) 3 domain, two from NS4 and one from NS5. Selected responses were shown to be HLA-A2 restricted, mediated by CD8+ T cells and to recognize endogenously synthesized viral antigen. Unexpectedly, peptide-specific CTL responses could also be induced in sero-negative individuals, suggesting in vitro activation of naive CTL precursors. The precursor frequency of peptide-specific CTL was 10 to 100-fold higher in infected patients compared to uninfected controls, and the responses were greatly diminished by removal of CD45 RO+ (memory) T cells. Further quantitative studies are clearly required to establish whether a correlation exists between the HCV-specific CTL response and the clinical course of this disease. Definition of the molecular targets of the human CTL response to HCV creates this opportunity, and may also contribute to the development of a T cell-based HCV vaccine.

HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release.

Cytotoxic T lymphocytes (CTL) are important to the control of viral replication and their presence may be important to disease outcome. An understanding of the spectrum of proteins recognized by hepatitis C virus (HCV)-specific CTL and the functional properties of these cells is an important step in understanding the disease process and the mechanisms of persistent infection, which occurs in the majority of HCV-infected individuals. In this report we identify HCV-specific CTL responses restricted by the HLA class I molecules A2, A3, A11, A23, B7, B8, and B53. The epitopes recognized by these intrahepatic CTL conform to published motifs for binding to HLA class I molecules, although in some cases we have identified CTL epitopes for which no published motif exists. The use of vectors expressing two different strains of HCV, HCV-1 and HCV-H, revealed both strain-specific and cross-reactive CTL. These HCV-specific CTL were shown to produce cytokines including IFN-gamma, TNF-alpha, GM-CSF, IL-8, and IL-10 in an antigen- and HLA class I-specific manner. These studies indicate that the CTL response to HCV is broadly directed and that as many as five different epitopes may be targeted in a single individual. The identification of minimal epitopes may facilitate peptide-specific immunization strategies. In addition, the release of proinflammatory cytokines by these cells may contribute to the pathogenesis of HCV-induced liver damage.

Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response.

The anti-viral T cell response is believed to play a central role in the pathogenesis of hepatitis C virus infection. Since chronic evolution occurs in > 50% of HCV infections, the sequential analysis of the T cell response from the early clinical stages of disease may contribute to define the features of the T cell response associated with recovery or chronic viral persistence. For this purpose, 21 subjects with acute hepatitis C virus infection were sequentially followed for an average time of 44 wk. Twelve patients normalized transaminase values that remained normal throughout the follow-up period; all but two cleared hepatitis C virus-RNA from serum. The remaining nine patients showed persistent viremia and elevated transaminases. Analysis of the peripheral blood T cell proliferative response to core, E1, E2, NS3, NS4, and NS5 recombinant antigens and synthetic peptides showed that responses to all hepatitis C virus antigens, except E1, were significantly more vigorous and more frequently detectable in patients who normalized transaminase levels than in those who did not. By sequential evaluation of the T cell response, a difference between the two groups of patients was already detectable at the very early stages of acute infection and then maintained throughout the follow-up period. The results suggest that the vigor of the T cell response during the early stages of infection may be a critical determinant of disease resolution and control of infection.

Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes.

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.

Role of hepatitis C virus in non-B chronic liver disease.

To assess the contribution of the recently identified hepatitis C virus to chronic liver diseases of unknown cause and chronic hepatitis attributed by exclusion to non-A, non-B hepatitis, we tested for antibody to hepatitis C in hepatitis B surface antigen-negative patients with a spectrum of chronic liver diseases. Antibody to hepatitis C virus, a marker of hepatitis C infection, was detected with a first-generation radioimmunoassay at the following frequencies in the following patient groups: 69% of transfusion-associated non-A, non-B hepatitis; 53% of non-transfusion-associated non-A, non-B hepatitis; 26% of hepatitis B surface antigen-negative hepatocellular carcinoma; 8% of cryptogenic cirrhosis; 5% to 7% of autoimmune chronic liver diseases; 19% of patients with miscellaneous types of chronic liver disease; and 0.67% of healthy controls. Among non-transfusion-associated cases, 81% with a history of intravenous drug use but only 18% with occupational exposure as health workers had antibody to hepatitis C virus. Among cases of hepatocellular carcinoma, 63% of Japanese patients but only 11% of American patients had evidence of hepatitis C infection. Comparison in a subgroup of 79 serum samples of a second-generation radioimmunoassay with the first-generation assay demonstrated a 12% increase in antibody frequency from 30% to 42%. We conclude that hepatitis C plays a substantial role in transfusion-associated and non-transfusion-associated non-A, non-B hepatitis as well as in hepatocellular carcinoma, especially in Japan, a limited role in cryptogenic cirrhosis, and essentially no role in autoimmune chronic liver diseases. Application of more sensitive immunoassays will increase the frequency of antibody seropositivity in all subgroups, but relative distinctions among risk groups are likely to remain.

Hepatitis C viral cDNA clones isolated from a healthy carrier donor implicated in post-transfusion non-A, non-B hepatitis.

Using a specific hepatitis C virus (HCV) antibody assay, positive blood donors responsible for the transmission of post-transfusion non-A, non-B hepatitis (PT-NANBH) were identified. cDNA fragments were isolated from one of the plasma samples of such healthy HCV carriers by using polymerase chain reactions. Nucleotide (nt) sequence analyses of the cDNA from three different regions of the viral genome revealed that they were derived from a Japanese HCV isolate that was similar but not identical to the prototype HCV previously isolated from a chronically infected chimpanzee. Homology at the nt and amino acid levels was comparatively lower in the presumed structural region than in putative nonstructural regions. This result not only confirms that HCV antibody-positive blood contains infectious HCV, but suggests the existence of different type(s) of HCV.

Prospects for a hepatitis C virus vaccine.

The scientific and clinical challenges that must be addressed and overcome in developing an efficacious HCV vaccine are substantial but not insurmountable. In a short period, considerable progress has been made in the understanding of HCV pathogenesis, epidemiology, and immunology, and the field of vaccinology in general is making very significant strides in developing new ways to activate and modulate immune responses. Advances in DNA vaccines, novel adjuvants, and recombinant protein technology may be keys in developing creative strategies to generate protective immunity against HCV.

Comparison of secretion of a hepatitis C virus glycoprotein in Saccharomyces cerevisiae and Kluyveromyces lactis.

A C-terminally truncated form of the hepatitis C virus (HCV) putative envelope glycoprotein E2 was expressed in two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, using a yeast signal peptide sequence to direct the viral glycoprotein to the endoplasmic reticulum (ER) pathway of secretion. Characterization of secreted E2 showed that the protein is endoglycosidase-H-sensitive in both yeasts. Moreover, in vivo inhibition of glycosylation with tunicamycin prevented secretion of E2 and showed that, of its 11 putative N-linked glycosylation sites, at least eight were core-glycosylated. Analysis of the heterologous glycoprotein by SDS-PAGE under nonreducing conditions and by gel filtration demonstrated the formation of multiple disulphides, which resulted in secretion of heterogeneous aggregates with an average molecular mass of 770-1000 kDa in both yeasts. However, variations were observed in the binding of the glycoprotein secreted by the two yeasts to a mannose-specific lectin, and also in its reactivity with anti-E2-specific antibodies. This denotes differences between the two yeasts in folding and/or modification of the E2 glycoprotein.

Effect of IL-12 on T-cell immune responses in patients with chronic HCV infection.

As the host's immune responses may determine the outcome of hepatitis C virus (HCV) infection, and interleukin (IL)-12 plays an essential role in host defense against infectious diseases, we studied the antigen-specific and non-specific cellular immune responses in patients with chronic HCV infection. A proliferative response to phytohemagglutinin (PHA) was found in all 20 patients. Of the 20, 8 (40%) displayed a lymphocyte proliferation in response to HCV antigen c22, 2 (10%) to c33, 6 (30%) to c100-3, and 1 (5%) to NS5. The addition of rhIL-12 to cultures of peripheral blood mononuclear cells (PBMC) stimulated with PHA significantly enhanced the proliferative responses in normal controls as well as in HCV-infected subjects. The increased proliferation was also observed in HCV-infected patients when PBMC were co-cultured with HCV antigens c22 and c100-3 in the presence of rhIL-12. The production of interferon gamma (IFNgamma), IL-2, IL-4 and IL-10 was observed in 7 (58.3%), 5 (41.7%), 3 (25.0%) and 5 (41.7%) HCV-infected individuals stimulated with c22, and in 4 (33.3%), 2 (16.7%), 2 (16.7%) and 2 (16.7%) with c100-3, respectively. All HCV-infected individuals had increased production of cytokines IFNgamma, IL-2, IL-4 and IL-10 in supernatants of PBMC after stimulation with PHA. IL-12 significantly augmented Th1 cytokine production in HCV-infected individuals stimulated with PHA and with HCV antigens. In conclusion, deficient cellular immune responses are present in HCV-infected patients and IL-12 can enhance the immune responses in these patients.

Scanning electron microscopy of chronically stimulated platinum intracochlear electrodes.

Platinum electrodes were examined for evidence of corrosion using a scanning electron microscope (SEM). In vivo electrodes, stimulated using charge-balanced biphasic pulses for periods of up to 2000 h at charge densities of 0.18-0.32 microC mm-2 geom. per phase, were compared with in vitro electrodes stimulated in inorganic saline using similar stimulus parameters, and with in vivo control electrodes. The in vitro stimulated electrodes showed evidence of platinum corrosion at high charge density and aggregate charge injection. Significantly, the in vivo stimulated electrodes showed no evidence of stimulus induced corrosion. Indeed, their surfaces were similar to the in vivo control electrodes. In vitro electrochemical studies have demonstrated that proteins play a significant role in the inhibition of platinum dissolution: the present study has demonstrated an inhibitory effect in vivo. This may be due to the presence of proteins.

Hearing loss prevalence and management in nursing home residents.

OBJECTIVES: To determine the prevalence and management of hearing loss and hearing handicap among non-demented nursing home residents. DESIGN: Descriptive study of total population of two nursing homes. PARTICIPANTS: All 121 eligible residents. SETTINGS: Two nursing homes. MINI OUTCOME MEASURES: Audiometry, questionnaires of nurses and subjects regarding perceived hearing handicap, and documentation in medical records of hearing loss. RESULTS: 77% had at least a mild (greater than or equal to 26 dB Hearing Level) hearing loss in the better ear, and 51% had a moderate to severe loss (greater than or equal to 41 dB HL). Self-assessments of hearing handicap by residents, together with audiometric findings and expressed interest in a hearing aid, were more useful guides for aural rehabilitation needs than were nurses' assessments of residents' handicaps. Medical records failed to identify 48% of residents with moderate to severe hearing losses. Fifty-eight percent of residents with severe hearing loss currently had hearing aids, and 38% more would like to have an aid. CONCLUSIONS: Residents should have hearing evaluations with documentation of results on admission and periodically under the direction of a nurse trained as a hearing specialist. Environmental modifications of the NH plus use of hearing aids can improve hearing.

Hepatitis C virus heteroduplex tracking assay : application to genotype determination, quasispecies analysis, and molecular evolution studies.

The heteroduplex tracking assay (HTA) is a tool that can be used for determining genotype, quasispecies analysis, molecular evolution, and epidemiological studies (1-7). By hybridizing a labeled, single-stranded DNA probe to colinear, reverse transcriptase (RT) PCR products from a sample of interest, the probe will either form a homoduplex with identical molecules or a heteroduplex with nonidentical sequences. The hybridization products are separated on MDE or polyacrylamide gels and visualized. Delwart et al., the developers of the HTA technique (1,2,7), have previously shown that the migration of heteroduplexes relative to the homoduplex on gels are approximately proportional to the percent nucleotide divergence between two species, and therefore, the genetic distance between two species can be determined. Genetic rearrangements, deletions, and/or insertions can alter the migration of heteroduplexes in a manner that disturbs the direct relationship between relative migration and genetic distance. Typically, heteroduplexes of 0.176-1.8 kb containing >1.4-3% to ~30% nucleotide substitutions, which lack genetic alterations, can be identified as unique species on MDE gels (1,4,6). The number and distribution of unique bands indicates the genetic complexity of viral species in each sample.

Psychiatric kibitzing.

Foreword from Dr. Sharfstein: In this era of managed care, utilization review, and concerns about costs, a clinician-reviewer who knows little about a case may make a decision that profoundly affects the course of a patient's treatment. Although Dr. Houghton's timely essay does not involve a managed care or utilization reviewer, it serves to remind us about the dangers inherent in criticizing the work and the judgment of other clinicians.