Nonstructural Antigens Research Articles

HCV-Specific T-Cell Response in Relation to Viral Kinetics and Treatment Outcome (DITTO-HCV Project)

The second slope of viral decline induced by interferon treatment has been suggested to be influenced mainly by the hepatitis C virus (HCV)-specific T-cell response; however, this hypothesis needs to be validated by results derived from experimental studies. To address this issue, the HCV-specific T-cell response of 32 genotype-1-infected patients of the 270 patients enrolled in the dynamically individualized treatment of hepatitis C infection and correlates of viral/host dynamics phase III, open-label, randomized, multicenter trial was studied in relation to viral kinetics and treatment outcome. Greater proliferative responses by HCV-specific CD8 cells were found before treatment in patients with a fast viral decline and with a sustained viral response. However, no significant improvement of HCV-specific CD8 responses was observed in the first weeks of therapy in both rapid viral responder and non-rapid viral responder patients. A mild enhancement of proliferative T-cell responses and a partial restoration of the cytotoxic T-cell potential was expressed only late during treatment, likely favored by HCV clearance. Early restoration of an efficient T-cell response does not seem to be an essential requirement for a rapid viral decline in the first weeks of treatment. However, patients presenting a better HCV-specific CD8 cell proliferative potential at baseline are more likely to present a rapid and sustained viral response. Therefore, future treatment protocols should consider the development of strategies aimed at improving HCV-specific T-cell responses.

Vaccines against human papillomavirus

Human papillomavirus has been identified as an etiological factor for cervical cancer, anogenital cancers and a subset of head and neck cancers. These important observations suggest that HPV vaccines have potential in the prevention and treatment of cervical cancer and other HPV-associated malignancies. The HPV genome encodes two HPV late genes, L1 and L2, which form the viral capsid. Early viral proteins support viral genome replication, two of which (E6 and E7) are important for HPV associated malignant transformation. Prophylactic HPV vaccines prevent infection by inducing neutralizing antibodies against HPV capsid proteins L1 and L2. However, because HPV-infected basal keratinocytes and HPV-transformed cells generally do not express L1 or L2, therapeutic HPV vaccines aim to treat established HPV infections and HPV-associated malignancies by targeting non-structural early viral antigens of HPV such as E6 and E7. Results from preclinical HPV vaccine studies have led to several HPV vaccine clinical trials. If these prophylactic and therapeutic HPV vaccines prove as successful in patients as they have in animal models, vaccination may provide for control and eventually eradication of oncogenic HPV infection and HPV-related cancers.

Pathogenesis of a Genogroup II Human Norovirus in Gnotobiotic Pigs

We evaluated the gnotobiotic (Gn) pig as a model to study the pathogenesis of human norovirus (HuNoV) and to determine the target cells for viral replication. Sixty-five Gn pigs were inoculated with fecal filtrates of the NoV/GII/4/HS66/2001/US strain or with pig-passaged intestinal contents (IC) and euthanized acutely (n = 43) or after convalescence (n = 22). Age-matched Gn piglets (n = 14) served as mock-inoculated controls. Seventy-four percent (48/65) of the inoculated animals developed mild diarrhea compared to 0 of 14 controls. Pigs from postinoculation days (PID) 1 to 4 tested positive for HuNoV by reverse transcription-PCR of rectal swab fluids (29/65) and IC (9/43) and by antigen (Ag) enzyme-linked immunosorbent assay (ELISA) using antiserum to virus-like particles of HuNoV GII/4. No control pigs were positive. Histopathologic examination showed mild lesions in the proximal small intestine of only one pig (1/7). Seroconversion after PID 21 was detected by antibody ELISA in 13 of 22 virus-inoculated pigs (titers, 1:20 to 1:200) but not in controls. Immunofluorescent microscopy using a monoclonal antibody to HuNoV GII capsid revealed patchy infection of duodenal and jejunal enterocytes of 18 of 31 HuNoV-inoculated pigs with a few stained cells in the ileum and no immunofluorescence (IF) in mock-inoculated controls. Immunofluorescent detection of the viral nonstructural N-terminal protein antigen in enterocytes confirmed translation. Transmission electron microscopy of intestines from HuNoV-inoculated pigs showed disrupted enterocytes, with cytoplasmic membrane vesicles containing calicivirus-like particles of 25 to 40 nm in diameter. In summary, serial passage of HuNoV in pigs, with occurrence of mild diarrhea and shedding, and immunofluorescent detection of the HuNoV structural and nonstructural proteins in enterocytes confirm HuNoV replication in Gn pigs.

Prospective study of viral clearance and CD4 + T-cell response in acute hepatitis C primary infection and reinfection

Background The outcome of acute hepatitis C is determined by early host–virus interactions, particularly involving the antiviral T-cell response. Objectives To identify early prognostic markers of spontaneous resolution of acute hepatitis C by performing a comprehensive analysis of viral and immunological factors during the natural course of acute HCV infection and reinfection. Study design 20 patients were investigated prospectively during acute HC or confirmed reinfection and 18 of them during follow up after spontaneous or treatment-induced elimination of the virus and resolution of the disease. Multiparameter flow cytometry was used to functionally characterize virus-specific CD4 + T-cell responses relative to the virologic outcome. Results Parallel immunologic and virologic monitoring of patients with acute HC identified distinct patterns of host–virus interaction related to HCV persistence or clearance. The highest frequency of antiviral Th1 cytokine-producing CD4 + T-cells was observed in patients with HCV reinfection, preceding rapid viral clearance within 3 weeks after disease onset. In all patients who subsequently cleared viremia, CD4 + T-cells produced Th1 cytokines following stimulation with non-structural HCV antigens (NS3 and NS4). In contrast, a chronic course of disease was associated with the absence of antiviral Th1 cytokine producing cells from the first weeks after onset of disease (acute persistent HC), or with fluctuating RNA levels (yo-yo pattern) and gradual waning of antiviral Th1 cells. Conclusions The results highlight the variability of immune response pattern in acute hepatitis C. Most importantly, “acute persistent hepatitis C” and a lack of TH1 effector cells within the first months of acute hepatitis C represent efficacious predictors of viral persistence and could thus be used as criteria in selecting candidates for early antiviral treatment.

P.284 Neurocognitive alterations in patients with HCV infection with and without abnormal transaminases

Background/Aims: To study whether the host's immune response determines viral clearance in chronic hepatitis C, virological markers and antigen-specific T cell reactions were analysed in 30 chronic HCV carriers followed up during interferon-α therapy, 11 untreated anti-HCV positive individuals and 10 healthy controls.Methods: Proliferative T helper cell responses to recombinant HCV core and non-structural antigens were monitored by 3H-thymidine uptake assay and compared to quantitative viraemia levels and HCV genotypes.Results: Of the 30 treated patients, six had sustained complete responses (20%), another six were transient therapy responders (20%) and 18 were non-responders (60%). Viral clearance was associated with the HCV genotype 3 and low pretreatment viral load. In a substantial proportion and complete and transient therapy responders, increased NS3-, helicase- and NS4-antigen-specific T cell responses were observed during interferon-α therapy. In non-responders and in the later clinical courses of transient therapy responders, core and NS5-specific T cell responses dominated. In addition, 11 untreated anti-HCV antibody positive individuals were studied. Two HCV-RNA negative patients who might have recovered from HCV infection showed strong persistent lymphoproliferative responses to NS3, helicase and NS4 antigens, whereas seven of the nine viraemic patients reacted with HCV core or NS5 antigens.Conclusions: Interferon-α treatment enhances NS3-, helicase- and NS4-antigen-specific T helper cell responses in patients with viral clearance, whereas viral persistence was associated with increased T cell reactivities against core and NS5 antigens. Immunogenetical, immunological and virological factors that may influence differential T cell induction in chronic hepatitis C are discussed.

Host immune responses in hepatitis C virus clearance

The factors that determine the outcome of hepatitis C virus (HCV) infection are not fully understood. An increased and broadly targeted/multispecific T-cell response is thought to be paramount to a favourable outcome. Human leucocyte antigen (HLA) genes, in particular DRB1 and DQB1, are also reported to influence outcome of infection. We have previously demonstrated strong associations between DRB10101 and spontaneous viral clearance. The aim of the current study was to investigate HCV-specific T-cell response and the influence of DRB10101 in patients with long-term history of HCV clearance as compared to patients that developed persistent HCV infection. The proliferation of peripheral blood mononuclear cells stimulated with five non-structural and core HCV antigens and 20 synthesized HCV peptides, designed using T-cell epitope-predictive software, was determined by the incorporation of H-thymidine. Although HCV-specific T-cell responses were more frequently detected and a broader range of peptides were targeted in the viral clearance group, the magnitude and breadth of the responses were not significantly different to that in the viral persistence group. The magnitude and breadth of the T-cell response was significantly associated, however, with possession of DRB10101. Furthermore DRB10101 positive individuals with viral clearance had broader HCV-specific T-cell responses. These findings lend further credence to the importance of the host immune system to the outcome of HCV infection and provide a rationale for the role of DRB10101 in the resolution of HCV infection.

Vaccines Against Human Papillomavirus and Cervical Cancer: Promises and Challenges

Cervical cancer and precancerous lesions of the genital tract are major threats to the health of women worldwide. The introduction of screening tests to detect cervical cancer precursor lesions has reduced cervical cancer rates in the developed world, but not in developing countries. Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer and dysplasia. Thus, cervical cancer and other HPV-associated malignancies might be prevented or treated by HPV vaccines. Two vaccine strategies have been developed. First, prevention of HPV infection through induction of capsid-specific neutralizing antibodies has been studied in clinical trials. However, because the capsid proteins are not expressed at detectable levels by infected basal keratinocytes or in HPV-transformed cells, a second approach of developing therapeutic vaccines by targeting nonstructural early viral antigens has also been developed. Because two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation and are coexpressed in the majority of HPV-containing carcinomas, most therapeutic vaccines target one or both of these gene products. A variety of approaches is being tested in therapeutic vaccine clinical trials, whereby E6 and/or E7 are administered in live vectors, as peptides or protein, in nucleic acid form, or in cell-based vaccines. The paradigm of preventing HPV infection through vaccination has been tested, and two vaccines are currently in phase III clinical trials. However, current therapeutic vaccine trials are less mature with respect to disease clearance. A number of approaches have shown significant therapeutic benefit in preclinical papillomavirus models and await testing in patient populations to determine the most effective curative strategy.

Quantitative analysis of anti–hepatitis C virus antibody–secreting B cells in patients with chronic hepatitis C†

To investigate the quantitative characteristics of humoral immunity in patients with hepatitis C, we established an enzyme-linked immunosorbent spot (ELISpot) assay for detection of anti-hepatitis C virus (HCV)-secreting B cells. Receiver operating characteristic curve analysis demonstrated 100% specificity and 58% to 92% sensitivity for detecting B-cell responses to NS5b, NS3, E2, and core antigens. The median sum of anti-HCV-secreting B cells to all HCV antigens tested was significantly higher in 39 patients with chronic hepatitis C (47.3 spot forming cells [SFCs]/10(6) peripheral blood mononuclear cells [PBMCs]) than in 9 recovered subjects (15.3 SFCs/10(6) PBMCs; P = .05) or 11 uninfected controls (5.3 SFCs/10(6) PBMCs; P < .001); the significant difference (P = .018) in chronic versus recovered patients was in reactivity to nonstructural antigens NS3 and NS5b. Anti-HCV immunoglubulin M (IgM)-secreting B cells were also readily detected and persisted decades into HCV infection; there was no difference in IgM-positive cells between chronic and recovered patients. ELISpot reactivity to genotype 1-derived antigens was equivalent in patients of genotypes 1, 2, and 3. There was significant correlation between the numbers of anti-HCV IgG-secreting B cells and serum aminotransferase and to the level of circulating antibody. In conclusion, ELISpot assays can be adapted to study B-cell as well as T-cell responses to HCV. Measurement at the single-cell level suggests that humoral immunity plays a minor role in recovery from HCV infection and that B-cell immunity is strongest in those with persistent infection.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Quantifying Antibodies to Japanese Encephalitis Virus Nonstructural 1 Protein To Detect Subclinical Infections in Vaccinated Horses

Antibodies to Japanese encephalitis virus (JEV) nonstructural 1 (NS1) protein constitute a marker of natural JEV infection among populations vaccinated with inactivated JE vaccine. In Japan, with few recent human JE cases, the natural infection rate is critical to evaluate the necessity of continuing JE vaccination. A sensitive immunochemical staining method for detecting NS1 antibodies in individuals naturally and subclinically infected with JEV was previously established. Here, an enzyme-linked immunosorbent assay (ELISA) to detect NS1 antibodies in equine sera was developed and evaluated as an alternative to immunostaining. By this method, NS1 antigens contained in culture fluids from cells stably transfected with the NS1 and NS2A genes were captured by a rabbit anti-NS1 polyclonal antibody. Three nanograms per well of NS1 antigen, corresponding to 1:2 to 1:8 dilutions of the culture fluid, was sufficient for testing. ELISA values were obtained by a single-serum dilution (1:100), which correlated with ELISA titers obtained by an endpoint method. Under a tentative cutoff value (0.122) statistically calculated from NS1 antibody levels of horses in an area where JEV is not endemic, a high level of qualitative agreement (85.3%) was obtained between the ELISA and immunostaining methods. A significant correlation coefficient (0.799; P < 0.001) was also obtained between the two methods. Three experimentally infected horses seroconverted no later than 13 to 23 days postinfection, whereas 4 field horses infected during an epizootic remained positive for NS1 antibodies for at least 40 weeks. Our results indicate that the ELISA used here was sufficiently sensitive to detect subclinical infections in vaccinated equine populations.

Vaccination to prevent and treat cervical cancer

Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer. Thus, cervical cancer and other HPV-associated malignancies might be prevented or treated by HPV vaccines. Transmission of papillomavirus may be prevented by the generation of antibodies to capsid proteins L1 and L2 that neutralize viral infection. However, because the capsid proteins are not expressed at detectable levels by infected basal keratinocytes or in HPV-transformed cells, therapeutic vaccines generally target nonstructural early viral antigens. Two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation and are coexpressed in the majority of HPV-containing carcinomas. Thus, therapeutic vaccines targeting E6 and E7 may provide the best option for controlling HPV-associated malignancies. Various candidate therapeutic HPV vaccines are currently being tested whereby E6 and/or E7 are administered in live vectors, as peptides or protein, in nucleic acid form, as components of chimeric virus-like particles, or in cell-based vaccines. Encouraging results from experimental vaccination systems in animal models have led to several prophylactic and therapeutic vaccine clinical trials. If these preventive and therapeutic HPV vaccines prove successful in patients, as they have in animal models, then oncogenic HPV infection and its associated malignancies may be controllable by vaccination.

Expansion of host-cell tropism of foot-and-mouth disease virus despite replication in a constant environment.

Foot-and-mouth disease virus (FMDV) variants adapted to BHK-21 cells showed an expanded host-cell tropism that extended to primate and human cell lines. Virus replication in human HeLa and Jurkat cells has been documented by titration of virus infectivity, quantification of virus RNA, expression of a virus-specific non-structural antigen, and serial passage of virus in the cells. Parallel serial infections of human Jurkat cells with the same variant FMDVs indicates a strong stochastic component in the progression of infection. Chimeric viruses identified the capsid as a genomic region involved in tropism expansion. These results indicate that, contrary to theoretical predictions, replication of an RNA virus in a constant cellular environment may lead to expansion of cellular tropism, rather than to a more specialized infection of the cellular type to which the virus has been adapted.

Sub-genomic replicons of Tick-borne encephalitis virus.

We constructed three sub-genomic replicons of Tick-borne encephalitis virus (TBEV) (Oshima REP, Oshima REP-GFP and Oshima REP-Neo) by deleting genes coding for structural proteins without or with insertion of green fluorescent protein (GFP) or Neo genes, respectively. BHK cells transfected with Oshima REP expressed the viral non-structural antigens in immunofluorescent and western blot analyses. GFP and viral antigens were co-expressed in the transfected cells with Oshima REP-GFP. G418-resistant cells harboring Oshima REP-Neo consistently expressed the antigens without showing any apparent CPE. These replicons constructed in this study will be useful in studies on the replication, assembly and packaging of TBEV, and to develop vaccines and gene-delivering systems.

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-gamma), protein-specific lymphoproliferative responses, IFN-gamma, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections.

Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (n = 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondissociated samples, 22 of these patients were found to be positive by the DBI. In the presence of DEN-specific immunoglobulin M antibodies, both viral RNA and DEN (NS-1) antigen could be detected. The number of positive samples identified by RT-PCR and DBI from these patients with primary DEN infections varied between 28 and 78%. In secondary DEN infections, the number of samples that tested positive by the DBI after immune-complex dissociation (DIS-DBI) was 25% higher than the number of samples that tested positive by RT-PCR and was 35% higher than that determined by nondissociated antigen (NDIS-DBI) detection. We conclude that the denKEY kit has limited diagnostic value for acute DEN infections compared to the RT-PCR and the NDIS-DBI and DIS-DBI methods. We clearly demonstrate that in secondary DEN infections the dissociation of NS-1 immune complexes is essential for early diagnosis of DEN infections.

Development of HPV vaccines for HPV-associated head and neck squamous cell carcinoma.

High-risk genotypes of the human papillomavirus (HPV), particularly HPV type 16, are found in a distinct subset of head and neck squamous cell carcinomas (HNSCC). Thus, these HPV-associated HNSCC may be prevented or treated by vaccines designed to induce appropriate HPV virus-specific immune responses. Infection by HPV may be prevented by neutralizing antibodies specific for the viral capsid proteins. In clinical trials, vaccines comprised of HPV virus-like particles (VLPs) have shown great promise as prophylactic HPV vaccines. However, given that capsid proteins are not expressed at detectable levels by infected basal keratinocytes, vaccines with therapeutic potential must target other non-structural viral antigens. Two HPV oncogenic proteins, E6 and E7, are important in the induction and maintenance of cellular transformation and are co-expressed in the majority of HPV-containing carcinomas. Therefore, therapeutic vaccines targeting these proteins may have potential to control HPV-associated malignancies. Various candidate therapeutic HPV vaccines are currently being tested whereby E6 and/or E7 is administered in live vectors, in peptides or protein, in nucleic acid form, as components of chimeric VLPs, or in cell-based vaccines. Encouraging results from experimental vaccination systems in animal models have led to several prophylactic and therapeutic vaccine clinical trials. Should they fulfill their promise, these vaccines may prevent HPV infection or control its potentially life-threatening consequences in humans.

Neutralizing antibodies in hepatitis c virus infection: a review of immunological and clinical characteristics

Chronic hepatitis C virus (HCV) infection is the most common cause of chronic liver disease in the United States, affecting more than 2.7 million people.1 Most cases of acute HCV result in chronic infection despite the appearance of antibodies to both structural and nonstructural HCV antigens. Hepatitis C replicates rapidly, at an estimated 1012 virions per day,2 and has a high mutation rate of approximately 10−3 nucleotide substitutions per genome site per year.3 The host humoral immune response contributes to the generation of viral diversity.

Influence of ethnicity in the outcome of hepatitis C virus infection and cellular immune response

This study was performed to examine the immunologic basis for the apparent ethnic difference in clinical outcome of hepatitis C virus (HCV) infection between African Americans (AA) and Caucasian Americans (CA). To this end, we recruited 99 chronically HCV-infected and 31 spontaneously HCV-cleared subjects for clinical, virologic, and immunologic analysis. In particular, CD4-proliferative T-cell response to genotype 1–derived HCV antigens (core, NS3-NS5) was examined in 82 patients chronically infected with genotype 1 (54 AA, 28 CA) and in all HCV-cleared subjects (14 AA, 17 CA). HCV-specific Th1 response also was examined in 52 chronic and 13 recovered subjects. Our results showed that HCV clearance was associated with a vigorous HCV-specific Th1 response irrespective of ethnic origin. Although the HCV-specific CD4 T-cell response clearly was weaker during chronic infection, AA ethnicity in this setting was associated with a significantly greater CD4-proliferative T-cell response to HCV, particularly to the nonstructural antigens (22% AA vs. 0% CA, P = .007) as well as better clinical parameters of liver disease. Interestingly, most HCV-specific CD4 T-cell proliferative responses in AA patients were unaccompanied by concurrent interferon γ (IFN-γ) production, suggesting a dysregulated virus-specific, CD4 T-cell effector function during chronic HCV infection. In conclusion, our results suggest that host ethnicity does influence the clinical outcome and antiviral T-cell response during HCV infection. AA ethnicity is associated with a more robust antiviral CD4 T-cell response than CA ethnicity, although these T cells are limited in direct virus or disease control due to their dysfunctional nature. (H EPATOLOGY 2003;37:590-599.)

A Novel Antigen Detection Immunoassay for Field Diagnosis of Hepatitis C Virus Infection

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (˜5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.

Frequencies of HCV-specific effector CD4+ T cells by flow cytometry: Correlation with clinical disease stages

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, affecting approximately 2% of the world's population. The immune mechanisms responsible for the highly variable natural history in a given individual are unknown. We used a multiparameter flow cytometric technique to functionally and phenotypically characterize HCV-specific effector T cells in the peripheral blood of 32 individuals with different stages of hepatitis C disease (resolved, mild chronic, advanced chronic) and normal controls. We found the highest frequencies of virus-specific effector cells with an activated memory phenotype (CD45RO+CD69+) in subjects who had resolved HCV infection, either spontaneously or with antiviral therapy. Effector cells from patients with resolved infection produced Th1 type cytokines following stimulation with nonstructural antigens (NS3 and NS4), whereas effector cells from chronically infected patients produced Th1 type cytokines predominantly following stimulation with the HCV core antigen. Stimulation with superantigen staphylococcal enterotoxin (SEB) induced the same levels of cytokine production in the different patient groups. Among the HCV-seropositive patients, viral load inversely correlated with the Th1 effector cell response to NS3. Interleukin (IL)-4 was produced only in response to the control antigens, but not in response to the HCV recombinant proteins. Taken together, these findings suggest that a vigorous HCV-specific CD4+ Th1 response, particularly against the nonstructural proteins of the virus, may be associated with viral clearance and protection from disease progression. Prospective studies using this new flow cytometric assay will be required to determine whether antiviral therapy modifies the frequency, specificity, and function of these virus-specific effector cells. (H EPATOLOGY 2002;35:190-198.)

Preventive and therapeutic vaccines for human papillomavirus-associated cervical cancers.

'High risk' genotypes of the human papillomavirus (HPV), particularly HPV type 16, are the primary etiologic agent of cervical cancer. Thus, HPV-associated cervical malignancies might be prevented or treated by induction of the appropriate virus-specific immune responses in patients. Sexual transmission of HPV may be prevented by the generation of neutralizing antibodies that are specific for the virus capsid. In ongoing clinical trials, HPV virus-like particles (VLPs) show great promise as prophylactic HPV vaccines. Since the capsid proteins are not expressed at detectable levels by basal keratinocytes, therapeutic vaccines generally target other nonstructural viral antigens. Two HPV oncogenic proteins, E6 and E7, are important in the induction and maintenance of cellular transformation and are coexpressed in the majority of HPV-containing carcinomas. Therefore, therapeutic vaccines targeting these proteins may provide an opportunity to control HPV-associated malignancies. Various candidate therapeutic HPV vaccines are currently being tested whereby E6 and/or E7 are administered in live vectors, in peptides or protein, in nucleic acid form, as components of chimeric VLPs, or in cell-based vaccines. Encouraging results from experimental vaccination systems in animal models have led to several prophylactic and therapeutic vaccine clinical trials. Should they fulfill their promise, these vaccines may prevent HPV infection or control its potentially life-threatening consequences in humans.