Evaluation of a new point-of-care oral anti-HCV test for screening of hepatitis C virus infection.

BACKGROUND: Hepatitis C virus (HCV) infection is a public health issue for which an effective universal screening method is urgently needed. An oral anti-HCV test could provide a noninvasive and rapid screening strategy for HCV infection. This study evaluated the performance of a new point-of-care oral assay developed by Well for the detection of HCV antibody. METHODS: Individuals from three centers with and without HCV infection were enrolled. All participants were tested for oral HCV antibody using the Well assay and for serum HCV antibody using established tests (ARCHITECT i2000 anti-HCV assay and InTec serum anti-HCV assay). For participants who obtained positive results, HCV RNA was tested for verification. Some patients underwent the OraQuick HCV test at the same time, and some self-tested with the Well assay during the same period. RESULTS: A total of 1179 participants, including 486 patients with chronic HCV infection, 108 patients with other liver diseases, and 585 individuals who underwent physical examination, were enrolled. The Well anti-HCV test had a sensitivity of 91.88% (95% confidence interval [CI]: 88.97-94.09%) and a specificity of 98.00% (96.58-98.86%) for oral HCV antibody detection. The consistency between the Well and InTec assays was 97.02% (1138/1179). The consistency between the Well and OraQuick assays was 98.50% (197/200). Furthermore, the results of self-testing were highly consistent with those of researcher-administered tests (Kappa = 0.979). In addition, the HCV RNA results also showed that HCV RNA could only be detected on 1 of the 39 false-negative samples, and for 172 positive HCV RNA results, 171 could be detected by the Well oral anti-HCV assay. CONCLUSIONS: The Well oral anti-HCV test offers high sensitivity and specificity and performed comparably to both the OraQuick assay and InTec assay for HCV diagnosis. Thus, the Well test represents a new tool for universal HCV screening to identify infected patients, particularly in regions with limited medical resources.

Inosine triphosphate pyrophosphatase polymorphisms are predictors of anemia in Chinese patients with chronic hepatitis C during therapy with ribavirin and interferon.

BACKGROUND AND AIM: Polymorphisms of inosine triphosphate pyrophosphatase (rs1127354 and rs6051702) and interferon lambda 4 (IFLN4) (rs12979860) are indicators of anemia and/or sustained virological response (SVR) in patients with chronic hepatitis C on ribavirin/interferon. The study aims to investigate the associations of rs1127354, rs6051702, and rs12979860 with hemoglobin levels and SVR in patients on ribavirin/interferon. METHODS: Polymorphisms were detected by pyrosequencing. Levels of hemoglobin and hepatitis C virus (HCV) RNA were measured at weeks 2, 4, 12, 24, 36, 48, and 72 of treatment. RESULTS: A total of 351 patients (median age, 50 years; male, 71.2%) were recruited and had HCV genotypes 1b (55.8%) or 2a (37.0%). Vedian baseline hemoglobin and HCV RNA were 155 g/dL and 6.07 log10 IU/mL. Major allele homozygosity was observed in 76.3% for rs1127354 (CC), 70.9% for rs6051702 (AA), and 89.7% for rs12979860 (CC). At 4 weeks of ribavirin/interferon treatment, a more significant reduction in hemoglobin was observed with rs112754 CC than with AC/AA (P < 0.05). A decline ≥3 g/dL was more common in patients with the rs112754 CC than with the other two polymorphisms. No significant change was observed regarding rs6051702 and rs12979860 variants. In the multivariable analysis, rs1127354 AA/AC (vs CC) were independently associated with lower odds of hemoglobin decline of > 3 g/dL at 4 weeks (odds ratio, 0.21; 95% CI, 0.09-0.46; P < 0.0001). In 258 patients with 72-week outcome data available, rs1127354, rs6051702, and rs12979860 were not associated with SVR (all P > 0.05). CONCLUSION: rs1127354 polymorphisms are associated with hemoglobin levels in Chinese patients with chronic hepatitis C treated with ribavirin/interferon.

Follicular helper T cell and memory B cell immunity in CHC patients.

Chronic hepatitis C (CHC) is associated with biological activity of T follicular helper (Tfh) cells and memory B cells (MBCs). However, the nature of Tfh cell subsets that are responsible for MBCs in CHC patients has not been evaluated. This study aimed to investigate Tfh and MBC immunity before and after direct-acting antiviral (DAA) therapy in patients with CHC. A total of 31 CHC patients and 15 healthy controls (HCs) were recruited. Individual patients were treated with sofosbuvir/ribavirin (SOF/RBV) or in combination with pegylated interferon alpha-2a (PEG-IFN-α-2a) for 12 weeks. Immunofluorescence revealed the frequency of ICOS+CD4+CXCR5+ active Tfh cells in liver tissue of CHC patients was higher than that of healthy control. Tfh and B cell co-culture experiments showed that Tfh2 cells from CHC patients have potential ability to induce B cell differentiation and IgG production. Flow cytometry showed that the frequencies of CD21-CD27+IgD- activated MBCs, ICOS+CD4+CXCR5+ activated Tfh cells, Tfh1 (IFN-γ+CD4+CXCR5+) cells, and Tfh2 (IL-4+CD4+CXCR5+) cells, but not of Tfh17 (IL-17+CD4+CXCR5+) cells, increased in CHC patients before and after DAA therapy. Collectively, ICOS+ Tfh, Tfh1, Tfh2 cells, and MBCs participated in the antiviral treatment process of SOF/RBV with or without PEG-IFN-α-2a in CHC patients, and their activity was further enhanced during the treatment. KEY MESSAGES: This study aimed to investigate Tfh cells and MBC immunity in CHC patients. CD21-CD27+IgD- activated MBCs increased in CHC patients before and after treatment. Tfh1 and Tfh2 cells increased in CHC patients before and after antiviral treatment. Intrahepatic activated Tfh cells increased in CHC patients before treatment. Tfh2 cells from CHC patients have a stronger ability to induce B cell differentiation.

Quantifying perinatal transmission of Hepatitis B viral quasispecies by tag linkage deep sequencing.

Despite full immunoprophylaxis, mother-to-child transmission (MTCT) of Hepatitis B Virus still occurs in approximately 2-5% of HBsAg positive mothers. Little is known about the bottleneck of HBV transmission and the evolution of viral quasispecies in the context of MTCT. Here we adopted a newly developed tag linkage deep sequencing method and analyzed the quasispecies of four MTCT pairs that broke through immunoprophylaxis. By assigning unique tags to individual viral sequences, we accurately reconstructed HBV haplotypes in a region of 836 bp, which contains the major immune epitopes and drug resistance mutations. The detection limit of minor viral haplotypes reached 0.1% for individual patient sample. Dominance of "a determinant" polymorphisms were observed in two children, which pre-existed as minor quasispecies in maternal samples. In all four pairs of MTCT samples, we consistently observed a significant overlap of viral haplotypes shared between mother and child. We also demonstrate that the data can be potentially useful to estimate the bottleneck effect during HBV MTCT, which provides information to optimize treatment for reducing the frequency of MTCT.

Comprehensive mapping of antigen specific T cell responses in hepatitis C virus infected patients with or without spontaneous viral clearance.

Elucidating protective immunity against HCV is important for the development of a preventative vaccine. We hypothesize that spontaneous resolution of acute HCV infection offers clue to protective immune responses, and that DAA therapy affects the quality and quantity of HCV-specific T cell responses. To test these hypotheses, we performed T cell epitope mapping in 111 HCV-infected individuals including 61 chronically HCV-1b (CHC-1b) infected, 24 chronically HCV-2a (CHC-2a) infected and 26 spontaneously recovered (SPR) patients with 376 overlapping peptides covering the entire HCV polyprotein. Selected T cell epitopes were then used to evaluate T cell responses in another 22 chronically HCV-1b infected patients on DAA therapy. Results showed that SPR had better HCV-specific T cell responses than CHC, as manifested by higher response rate, greater magnitude and broader epitope coverage. In addition, SPR recognized novel epitopes in Core, E1, E2, NS4B, NS5A regions that were not present in the CHC. Furthermore, during the first 24 weeks of DAA therapy, there was no functional immune reconstitution of HCV-specific T cells. These results indicate that T cell responses may be a correlate of protection. Therefore, effective preventative vaccines should elicit a robust T cell response. Although various DAA regimens efficiently cleared viruses from the blood of HCV-infected patients, there was no contemporaneous early T cell immune reconstitution, suggesting that early treatment is needed for preserving the functions of HCV-specific T cells.

Long-term effect on natural killer cells by interferon-α therapy on the outcomes of HCV infection.

Natural killer (NK) cells act as innate immune cells against hepatitis C virus (HCV) infection. Interferon-α (IFN-α) and ribavirin are the standard treatments for patients with HCV infection. This study is aimed at investigating the dynamic changes in the frequency of different subsets of NK cells following treatment in xx chronic hepatitis C (CHC) patients. CHC patients were treated with peg-IFN or IFN-α, and followed up for 72 weeks. The frequency of different subsets of NK in CHC patients was determined longitudinally by flow cytometry. Treatment with the standard therapy increased the percentages of NKp30(+), NKp46(+), and CD107a(+) NK cells, which were positively correlated with the declining of serum HCV-RNA, but not IFN-γ(+) NK cells. NKG2A(+) and KIR2DL3(+) NK cells were associated with an early virological response in CHC patients. Treatment with IFN-α adjusts the balance of activated receptors and inhibitory receptors and enhances the cytotoxic activity of NK cells. Therefore, measuring NK subsets may be valuable for therapeutic responses in CHC patients.

Antiviral treatment improves disrupted peripheral B lymphocyte homeostasis in chronic hepatitis B virus-infected patients.

Disruption of peripheral blood B-cell homeostasis and variation of surface receptors occur with certain infections and autoimmune diseases. However, the impact of antiviral therapy on B-cell alteration during chronic hepatitis B (CHB) infection remains unclear. Our study aims to document the effects of B-cell alteration in CHB patients treated with tenofovir or adefovir. A total of 21 CHB patients and 10 healthy donors were recruited into the study. We identified B-cell subsets by flow cytometry and observed changes in the B-cell repertoire of CHB patients upon tenofovir or adefovir antiviral treatment. The total and percent of B cells and CD5 + B-cell subsets were significantly increased in CHB patients compared to healthy donors. Total and percent of CD5 + B cells gradually decreased following the diminution of the HBV DNA load after tenofovir and adefovir treatment. Upon tenofovir treatment, the percent of memory CD27 + B cells was increased but the absolute number declined, whereas naïve CD27- B cells declined in both percent and absolute number. In the adefovir treatment group, neither naïve nor memory B cells were altered by the treatment. Furthermore, CHB patients displayed higher levels of activation markers (CD69 and CD24) and trended towards restored B-cell homeostasis after antiviral treatment. In conclusion, disrupted B-cell homeostasis is an important feature of CHB patients and is partially restored after control of viral replication by antiviral treatment. B-cell antiviral immunity is improved by restoring B-cell homeostasis and activation.

Antiviral treatment alters the frequency of activating and inhibitory receptor-expressing natural killer cells in chronic hepatitis B virus infected patients.

Natural killer (NK) cells play a critical role in innate antiviral immunity, but little is known about the impact of antiviral therapy on the frequency of NK cell subsets. To this aim, we performed this longitudinal study to examine the dynamic changes of the frequency of different subsets of NK cells in CHB patients after initiation of tenofovir or adefovir therapy. We found that NK cell numbers and subset distribution differ between CHB patients and normal subjects; furthermore, the association was found between ALT level and CD158b(+) NK cell in HBV patients. In tenofovir group, the frequency of NK cells increased during the treatment accompanied by downregulated expression of NKG2A and KIR2DL3. In adefovir group, NK cell numbers did not differ during the treatment, but also accompanied by downregulated expression of NKG2A and KIR2DL3. Our results demonstrate that treatment with tenofovir leads to viral load reduction, and correlated with NK cell frequencies in peripheral blood of chronic hepatitis B virus infection. In addition, treatments with both tenofovir and adefovir in chronic HBV infected patients induce a decrease of the frequency of inhibitory receptor(+) NK cells, which may account for the partial restoration of the function of NK cells in peripheral blood following treatment.

Enhancing the antihepatitis B virus immune response by adefovir dipivoxil and entecavir therapies.

Chronicity of hepatitis B (CHB) infection is characterized by a weak immune response to the virus. Entecavir (ETV) and adefovir dipivoxil (ADV) are effective in suppressing hepatitis B virus (HBV) replication. However, the underlying immune mechanism in the antiviral response of patients treated with nucleoside or nucleotide analogs is not clearly understood. In this study, regulatory T cells (Tregs) and intracellular cytokines, including IL-2, interferon (IFN)-γ, tumor-necrosis factor (TNF)-α and IL-4, were measured prior to and at 12, 24, 36 and 48 weeks after treatment with ETV or ADV. The cytokines were increased from 24 to 48 weeks after treatment. Higher levels of Th1 cytokines were observed with ETV (n=29) versus ADV (n=28) treatment. By contrast, the numbers of Tregs in both groups were decreased. The altered cytokine profile and cellular component was accompanied by a decrease in HBV DNA levels in both groups, which may contribute to their therapeutic effect in CHB infection. Our findings suggest that the antiviral effect of the drugs may be attributed not only to their direct effect on virus suppression but also to their immunoregulatory capabilities.

Th1 and Th2 immune response in chronic hepatitis B patients during a long-term treatment with adefovir dipivoxil.

Adefovir dipivoxil treatment has significantly improved the outcome of chronic hepatitis B virus (HBV) infection. However, it remains largely unknown how immune system responds to the treatment. Chronic HBV patients were treated with adefovir dipivoxil and examined for serum HBV DNA loads, cytokines, and T helper (Th1) and 2 (Th2) cytokine producing T cells during 104 weeks of the treatment. Th1/Th2 cytokines producing T cells were significantly lower in chronic HBV patients as compared to normal individuals. Adefovir dipivoxil treatment led to the increase of Th1/Th2 cytokines producing T cells and serum cytokine levels in association with the decline of HVB DNA load. In contrast, Th1/Th2 cytokines producing T cells remained lower in one patient detected with adefovir dipivoxil resistant HBV A181T/V mutation. This study has established inverse correlation of the increase of Th1/Th2 immunity and the decline of HBV DNA load in chronic HBV patients during adefovir dipivoxil treatment.