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1.
Viruses ; 11(7)2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31288455

RESUMO

BACKGROUND: To date, there is no licensed vaccine available to prevent respiratory syncytial virus (RSV) infection. The valuable pre-fusion conformation of the fusion protein (pre-F) is prone to lose high neutralizing antigenic sites. The goals of this study were to stabilize pre-F protein by fixatives and try to find the possibility of developing an inactivated RSV vaccine. METHODS: The screen of the optimal fixative condition was performed with flow cytometry. BALB/c mice were immunized intramuscularly with different immunogens. The serum neutralizing antibody titers of immunized mice were determined by neutralization assay. The protection and safety of these immunogens were assessed. RESULTS: Fixation in an optimal concentration of formaldehyde (0.0244%-0.0977%) or paraformaldehyde (0.0625%-1%) was able to stabilize pre-F. Additionally, BALB/c mice inoculated with optimally stabilized pre-F protein (opti-fixed) induced a higher anti-RSV neutralization (9.7 log2, mean value of dilution rate) than those inoculated with unstable (unfixed, 8.91 log2, p < 0.01) or excessively fixed (exce-fixed, 7.28 log2, p < 0.01) pre-F protein. Furthermore, the opti-fixed immunogen did not induce enhanced RSV disease. CONCLUSIONS: Only the proper concentration of fixatives could stabilize pre-F and the optimal formaldehyde condition provides a potential reference for development of an inactivated RSV vaccine.

2.
Nat Commun ; 10(1): 3192, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324803

RESUMO

Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.

3.
Sci China Life Sci ; 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31231780

RESUMO

A new HPV-16/18 bivalent vaccine expressed by the Escherichia coli has been proven to be efficacious in adult women. A randomized, immunogenicity noninferiority study of this candidate vaccine was conducted in December 2015 in China. Girls aged 9-14 years were randomized to receive 2 doses at months 0 and 6 (n=301) or 3 doses at months 0, 1 and 6 (n=304). Girls aged 15-17 years (n=149) and women aged 18-26 years (n=225) received 3 doses. The objectives included noninferiority analysis of the IgG geometric mean concentration (GMC) ratio (95% CI, lower bound>0.5) to HPV-16 and HPV-18 at month 7 in girls compared with women. In the per-protocol set, the GMC ratio of IgG was noninferior for girls aged 9-17 years receiving 3 doses compared with women (1.76 (95% CI, 1.56, 1.99) for HPV-16 and 1.93 (95% CI, 1.69, 2.21) for HPV-18) and noninferior for girls aged 9-14 years receiving 2 doses compared with women (1.45 (95% CI, 1.25, 1.62) for HPV-16 and 1.17 (95% CI, 1.02, 1.33) for HPV-18). Noninferiority was also demonstrated for neutralizing antibodies. The immunogenicity of the HPV vaccine in girls receiving 3 or 2 doses was noninferior compared with that in young adult women.

4.
Hepatology ; 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31121067

RESUMO

It is not clear whether baseline hepatitis B core antibody (anti-HBc) level in hepatitis B e antigen (HBeAg)-positive children with a normal alanine aminotransferase (ALT) level is predictive of spontaneous HBeAg seroconversion. We investigated the correlation between anti-HBc level and the natural course of chronic hepatitis B (CHB) virus (HBV) infection in children, particularly the ability of baseline anti-HBc level to predict spontaneous HBeAg seroconversion during long-term follow-up. HBeAg-positive children with untreated CHB and a normal ALT level were followed longitudinally. Anti-HBc level was determined by double-sandwich immunoassay. Effects of anti-HBc levels and other parameters on spontaneous HBeAg seroconversion and the natural course of CHB were assessed. A total of 182 children (106 males) with a median age at enrollment of 10.6 years (interquartile range [IQR], 10.3-15.3) were followed for a median of 19.8 years (IQR, 11.9-21.9). Spontaneous HBeAg seroconversion occurred in 85 children (46.7%) during the follow-up. A baseline anti-HBc titer of >500 IU/mL (hazard ratio [HR] = 2.81), HBV genotype B and B + C (HR = 3.46), and a baseline hepatitis B surface antigen titer of ≤4.8 log10  IU/mL (HR = 3.09) were predictive of spontaneous HBeAg seroconversion, based on multivariable survival analysis (P < 0.001). In cases remaining HBeAg positive, their anti-HBc levels increased gradually during follow-up because of ongoing inflammation. Conclusion: Baseline anti-HBc level is predictive of spontaneous HBeAg seroconversion in HBeAg-positive children with a normal ALT level. Anti-HBc level reflects anti-HBV immune response in the HBeAg-positive normal ALT phase of CHB.

5.
Emerg Microbes Infect ; 8(1): 724-733, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130075

RESUMO

Anti-HBs is a well-known marker of protective capability against HBV. However, little is known about the association between the qAnti-HBs determined by immunoassays and the neutralization activity (NAT) derived from functional assays. We developed an in vitro assay for direct measurement of the NAT of human sera. The new assay was highly sensitive, with an analytical sensitivity of 9.6 ± 1.3 mIU/mL for the HBIG standard. For serum detection, the maximum fold dilution required to produce ≥50% inhibition (MDF50) of HBV infection was used as the quantitative index. In vitro NAT evaluations were conducted for a cohort of 164 HBV-free healthy individuals. The results demonstrated that the NAT positively correlated with the qAnti-HBs (R2 = 0.473, p < 0.001). ROC analysis indicated that the optimal cutoff value of the qAnti-HBs to discriminate significant NAT (MDF50 ≥ 8) was 62.9 mIU/mL, with an AUROC of 0.920. Additionally, we found that the qAnti-HBc was another independent parameter positively associated with the NAT (R2 = 0.300, p < 0.001), which suggested that antibodies against other HBV proteins generated by previous HBV exposure possibly also contribute to the NAT. In summary, the new cell-based assay provides a robust tool to analyse the anti-HBV NAT. Abbreviations: HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; Anti-HBs: Hepatitis B surface antibody; HBeAg: Hepatitis B e antigen; Anti-HBc: Hepatitis B core antibody; qAnti-HBs: quantitative hepatitis B surface antibody; qAnti-HBc: quantitative hepatitis B core antibody; qHBeAg: quantitative hepatitis B e antigen; NAT: neutralization activity; HBIG: hepatitis B immune globulin; NTCP: Na+-taurocholate cotransporting polypeptide; IRES: internal ribosome entry site; ccHBV: cell culture derived hepatitis B virus; GE/cell: genome equivalent per cell; MOI: multiplicity of infection; Dpi: day post infection; HepG2-TetOn: a HepG2-derived cell line that expresses the doxycycline-regulated transactivator; ROC: receiver operating characteristic curve; AUROC: area under receiver operating characteristic curve; LLOQ: the lower limits of quantification; MDF50: the maximum fold dilution required to produce ≥50% inhibition; IC50: half maximal inhibitory concentration.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Anti-Hepatite B/sangue , Vírus da Hepatite B/imunologia , Testes de Neutralização/métodos , Soro/imunologia , Células Hep G2 , Humanos , Curva ROC , Sensibilidade e Especificidade
6.
J Natl Cancer Inst ; 2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31086947

RESUMO

Background The high cost and insufficient supply of human papillomavirus (HPV) vaccines have slowed the pace of controlling cervical cancer. A phase 3 clinical trial was conducted to evaluate the efficacy, safety and immunogenicity of a novel Escherichia coli-produced bivalent HPV-16/18 vaccine. Methods A multi-centre, randomized, double-blind trial started on November 22, 2012, in China. In total, 7372 eligible women aged 18-45 years were age-stratified and randomly assigned to receiving 3 doses of the test or control (hepatitis E) vaccine at months 0, 1 and 6. Co-primary endpoints included high-grade genital lesions and persistent infection (over 6 months) associated with HPV-16/18. The primary analysis was performed on a per-protocol susceptible population of individuals who were negative for relevant HPV type-specific neutralizing antibodies (at day 0) and DNA (at day 0 through month 7) and who received 3 doses of the vaccine. This report presents data from a pre-specified interim analysis used for regulatory submission. Results In the per-protocol cohort, the efficacies against high-grade genital lesions and persistent infection were 100.0% (95% confidence interval [CI] = 55.6% to 100.0%, 0/3306 in the vaccine group vs. 10/3296 in the control group) and 97.8% (95% CI = 87.1% to 99.9%, 1/3240 vs. 45/3246), respectively. The side effects were mild. No vaccine-related serious adverse events were noted. Robust antibody responses for both types were induced and persisted for at least 42 months. Conclusions The Escherichia coli-produced HPV-16/18 vaccine is well tolerated and highly efficacious against HPV-16/18 associated high-grade genital lesions and persistent infection in women.

7.
Gut ; 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926653

RESUMO

OBJECTIVE: This study aimed to develop a novel therapeutic vaccine based on a unique B cell epitope and investigate its therapeutic potential against chronic hepatitis B (CHB) in animal models. METHODS: A series of peptides and carrier proteins were evaluated in HBV-tolerant mice to obtain an optimised therapeutic molecule. The immunogenicity, therapeutic efficacy and mechanism of the candidate were investigated systematically. RESULTS: Among the HBsAg-aa119-125-containing peptides evaluated in this study, HBsAg-aa113-135 (SEQ13) exhibited the most striking therapeutic effects. A novel immunoenhanced virus-like particle carrier (CR-T3) derived from the roundleaf bat HBV core antigen (RBHBcAg) was created and used to display SEQ13, forming candidate molecule CR-T3-SEQ13. Multiple copies of SEQ13 displayed on the surface of this particulate antigen promote the induction of a potent anti-HBs antibody response in mice, rabbits and cynomolgus monkeys. Sera and purified polyclonal IgG from the immunised animals neutralised HBV infection in vitro and mediated efficient HBV/hepatitis B virus surface antigen (HBsAg) clearance in the mice. CR-T3-SEQ13-based vaccination induced long-term suppression of HBsAg and HBV DNA in HBV transgenic mice and eradicated the virus completely in hydrodynamic-based HBV carrier mice. The suppressive effects on HBsAg were strongly correlated with the anti-HBs level after vaccination, suggesting that the main mechanism of CR-T3-SEQ13 vaccination therapy was the induction of a SEQ13-specific antibody response that mediated HBV/HBsAg clearance. CONCLUSIONS: The novel particulate protein CR-T3-SEQ13 suppressed HBsAg effectively through induction of a humoural immune response in HBV-tolerant mice. This B cell epitope-based therapeutic vaccine may provide a novel immunotherapeutic agent against chronic HBV infection in humans.

8.
Clin Lab ; 65(3)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30868849

RESUMO

BACKGROUND: Nepal is an endemic area for hepatitis E virus (HEV) epidemics. The research on viral hepatitis in Nepal is limited. METHODS: Serum samples from 170 patients presenting with symptoms of hepatitis were collected from April to May 2014 in Biratnagar, Nepal, and then transported to Xiamen, China, for further evaluation. All samples were tested for HEV RNA, HEV antigen, anti-HEV IgM, anti-HEV IgG and anti-HBc IgM, anti-HCV IgG, and anti-HAV IgM. RESULTS: Sixteen patients were identified as acute hepatitis E with the presence of ≥ 2 HEV acute phase markers (antigen, RNA, and anti-HEV IgM). HEV infection was the major cause of potential active viral hepatitis (59.2%, 16 of 27), followed by HBV (25.9%, 7 of 27, anti-HBc IgM positive), HAV (18.5%, 5 of 27, anti-HAV IgM positive), and HCV (3.7%, 1 of 27, anti-HCV antibodies). All 16 confirmed HE cases were positive for HEV antigen, while 5 cases were HEV RNA positive, as well as 15 anti-HEV IgM positive. The low positive rate of RNA might be related to the collection and/or the transportation of these samples. CONCLUSIONS: This study showed that HEV is a major cause of acute hepatitis in developing countries and regions. Application of immunoassay diagnostic kits, especially the HEV antigen tests, showed great potential for HE detection in these countries and regions.


Assuntos
Países em Desenvolvimento , Vírus da Hepatite E/imunologia , Hepatite E/diagnóstico , Vírus da Hepatite E/genética , Humanos , Nepal
9.
J Cell Sci ; 132(5)2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30683797

RESUMO

The Caenorhabditis elegans aminophospholipid translocase TAT-1 maintains phosphatidylserine (PS) asymmetry in the plasma membrane and regulates endocytic transport. Despite these important functions, the structure-function relationship of this protein is poorly understood. Taking advantage of the tat-1 mutations identified by the C. elegans million mutation project, we investigated the effects of 16 single amino acid substitutions on the two functions of the TAT-1 protein. Two substitutions that alter a highly conserved PISL motif in the fourth transmembrane domain and a highly conserved DKTGT phosphorylation motif, respectively, disrupt both functions of TAT-1, leading to a vesicular gut defect and ectopic PS exposure on the cell surface, whereas most other substitutions across the TAT-1 protein, often predicted to be deleterious by bioinformatics programs, do not affect the functions of TAT-1. These results provide in vivo evidence for the importance of the PISL and DKTGT motifs in P4-type ATPases and improve our understanding of the structure-function relationship of TAT-1. Our study also provides an example of how the C. elegans million mutation project helps decipher the structure, functions, and mechanisms of action of important genes.

10.
Emerg Infect Dis ; 24(12): 2241-2250, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30457530

RESUMO

All hepatitis E virus (HEV) variants reported to infect humans belong to the species Orthohepevirus A (HEV-A). The zoonotic potential of the species Orthohepevirus C (HEV-C), which circulates in rats and is highly divergent from HEV-A, is unknown. We report a liver transplant recipient with hepatitis caused by HEV-C infection. We detected HEV-C RNA in multiple clinical samples and HEV-C antigen in the liver. The complete genome of the HEV-C isolate had 93.7% nt similarity to an HEV-C strain from Vietnam. The patient had preexisting HEV antibodies, which were not protective against HEV-C infection. Ribavirin was an effective treatment, resulting in resolution of hepatitis and clearance of HEV-C viremia. Testing for this zoonotic virus should be performed for immunocompromised and immunocompetent patients with unexplained hepatitis because routine hepatitis E diagnostic tests may miss HEV-C infection. HEV-C is also a potential threat to the blood product supply.

11.
Artigo em Inglês | MEDLINE | ID: mdl-30267868

RESUMO

For chronic hepatitis B patients, hepatitis B e antigen (HBeAg) seroclearance signals a transition from an immunologically active phase to an inactive carrier state with a reduction in hepatitis B virus (HBV) DNA levels and a reduced risk of hepatocellular carcinoma (HCC).1 Predictors of HBeAg seroclearance include lower HBV DNA levels, viral genotype, the precore mutation, and higher serum alanine aminotransferase (ALT) levels.2.

12.
J Virol Methods ; 260: 34-40, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30003925

RESUMO

A licensed vaccine for respiratory syncytial virus (RSV) has yet to be developed, and a reliable and repeatable neutralizing assay is indispensable for vaccine development. Here, we demonstrated an optimized high-throughput RSV neutralization assay that utilizes a fluorescence plate reader (reader) as a substitute for flow cytometry to detect fluorescent signals in RSV-A2 mKate-infected cells. Furthermore, this study tested the influence of virus input and infectivity on the neutralizing assay and highlighted critical factors (together with a suggested protocol) for obtaining stable data using this assay.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Ensaios de Triagem em Larga Escala , Testes de Neutralização , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Pré-Escolar , Fluorescência , Voluntários Saudáveis , Células Hep G2 , Humanos , Camundongos , Pessoa de Meia-Idade , Reprodutibilidade dos Testes
13.
Emerg Microbes Infect ; 7(1): 125, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29977038

RESUMO

Hepatitis E virus (HEV) is emerging as a potential threat to the safety of blood transfusions. In many countries and regions endemic for HEV, such as China, blood donors are not routinely tested for HEV infection. In this study, 11747 eligible blood donors were screened for anti-HEV immunoglobulin M (IgM)/immunoglobulin G (IgG) and HEV RNA and antigen in China. Twenty-four donors who were positive for both HEV antigen and RNA were followed for ≥ 70 days, and none of these donors reported clinical hepatitis or illness. At least 1 follow-up sample was provided by 17 donors, including 10 with viremia and/or antigenemia for ≥ 70 days and 3 with antigen and RNA positivity for >90 days. Fourteen of the 17 donors did not present with an obvious serologic response during the follow-up period. These results differed from previous reports, in which viremia lasted for 68 days and elicited an antibody response. These donors showed atypical HEV infection progression that differed from that of hepatitis E patients. The presence of these donors presents a challenge for transfusion transmission screening.


Assuntos
Doadores de Sangue , Seleção do Doador , Anticorpos Anti-Hepatite/sangue , Vírus da Hepatite E/patogenicidade , Hepatite E/sangue , RNA Viral/sangue , Soroconversão/fisiologia , Adulto , Biomarcadores/sangue , China/epidemiologia , Feminino , Hepatite E/epidemiologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Viremia , Adulto Jovem
14.
Artigo em Inglês | MEDLINE | ID: mdl-29753083

RESUMO

BACKGROUND & AIMS: Although a low level of hepatitis B surface antigen (HBsAg) is a marker of hepatitis B virus (HBV) seroclearance, additional biomarkers are needed for more accurate prediction. We investigated whether quantification of antibody against HBV core protein (anti-HBc) can identify patients with undetectable levels of HBV DNA and HBsAg seroclearance among those who were HBV e antigen (HBeAg)-seronegative. METHODS: We performed a retrospective analysis of data from a community-based cohort of individuals (30-65 years old) in Taiwan who were HBsAg seropositive, anti-HCV negative, and free of cirrhosis and or liver cancer, recruited from 1991 through 1992, and evaluated every 6-12 months until June 30, 2004. We measured levels of anti-HBc in blood samples from 2500 participants who were seronegative for HBeAg. The first date at which a sample tested negative for HBV DNA or HBsAg, and remained negative in subsequent tests, was designated as the date of spontaneous HBV DNA undetectability or HBsAg seroclearance. We calculated cumulative incidences of seroclearance; associations between level of anti-HBc and undetectability of HBV DNA or HBsAg seroclearance were estimated by Cox proportional hazard regression. The effects of time on the associations between level of anti-HBc and seroclearance was assessed by the area under the receiver operating characteristic curve (AUROC) analysis. RESULTS: After a 12-year follow-up period, higher proportions of subjects with levels of anti-HBc <3 log IU/mL had undetectable levels of HBV DNA (58%) and HBsAg seroclearance (53%) than subjects with higher levels of anti-HBc (29.6% and 19.8%, respectively) (P < .001). For subjects with levels of HBsAg <102 IU/mL and anti-HBc <3 log IU/mL, the adjusted rate ratio of HBV DNA undetectability was 16.45 (95% CI, 11.15-24.28) and of HBsAg seroclearance was 17.95 (95% CI, 12.49-25.81), compared to subjects with higher levels of HBsAg and anti-HBc. A model that included level of anti-HBc as a parameter identified subjects with HBsAg seroclearance within 10 years with an AUROC of 82%; this value was significantly higher than that from models that include only level of HBV DNA and HBsAg (P < .0001). CONCLUSIONS: In a retrospective analysis of a large cohort of patients with chronic HBV infection in Taiwan, we associated levels of anti-HBc <3 log IU/mL with undetectable HBV DNA and HBsAg seroclearance within 10 years; patients who also have levels of HBsAg <102 IU/mL have greater odds. Combining data on levels of HBsAg, HBV DNA, and anti-HBc identify HBeAg-seronegative patients with HBsAg seroclearance with AUROC value of 82%.

16.
Dig Liver Dis ; 50(5): 482-489, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29396134

RESUMO

BACKGROUNDS: The evaluation of liver fibrosis stages is essential for the clinical management of chronic hepatitis B (CHB). AIMS: To develop and validate a novel noninvasive index for moderate to severe fibrosis (≥S2) in CHB patients. METHODS: A total of 401 CHB patients who underwent liver biopsy were divided into the training (n = 300) and validation (n = 101) cohort. Histological severity was scored using a modified Scheuer system. Clinical and laboratory assessments were collected. RESULTS: In the training cohort, PACG, a novel index combining the quantitative hepatitis B core antibody (qAnti-HBc), platelet count (PLT), and albumin globulin ratio (A/G), presented better diagnostic performance (AUROC = 0.814) than that of APRI (0.735, p = 0.007) and FIB-4 (0.749, p = 0.014). In the validation cohort, the AUROC of the PACG, APRI, FIB-4 and Fibroscan were 0.834, 0.806, 0.791 and 0.810, respectively. More importantly, a higher and lower cutoff of PACG for predicting ≥S2 fibrosis or not had a >90% sensitivity and specificity, with a diagnostic accuracy of 85.9%. CONCLUSION: PACG is a promising noninvasive alternative to liver biopsy in CHB patients for the evaluation of moderate to severe fibrosis.


Assuntos
Globulinas/metabolismo , Anticorpos Anti-Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Hepatite B Crônica/complicações , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico , Albumina Sérica/metabolismo , Adulto , Fatores Etários , Alanina Transaminase/sangue , Área Sob a Curva , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Técnicas de Imagem por Elasticidade , Feminino , Humanos , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Masculino , Contagem de Plaquetas , Valor Preditivo dos Testes , Curva ROC , Estudos Retrospectivos , Índice de Gravidade de Doença
17.
Theranostics ; 8(2): 549-562, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29290826

RESUMO

Rationale: Monoclonal antibodies (mAbs) mostly targeting extracellular or cell surface molecules have been widely used in the treatment of various diseases. However, mAbs cannot pass through the cell membrane as efficiently as small compounds, thus limiting their use against intracellular targets. Methods to shuttle antibodies into living cells may largely expand research and application in areas based on mAbs. Hepatitis B virus X protein (HBx) is an important intracellular multi-functional viral protein in the life cycle of hepatitis B virus (HBV). HBx plays essential roles in virus infection and replication and is strongly associated with HBV-related carcinogenesis. Methods: In this study, we developed a cell-penetrating whole molecule antibody targeting HBx (9D11-Tat) by the fusion of a cell penetrating peptide (CPP) on the C-terminus of the heavy chain of a potent mAb specific to HBx (9D11). The anti-HBV effect and mechanism of 9D11-Tat were investigated in cell and mouse models mimicking chronic HBV infection. Results: Our results demonstrated that the recombinant 9D11-Tat antibody could efficiently internalize into living cells and significantly suppress viral transcription, replication, and protein production both in vitro and in vivo. Further analyses suggested the internalized 9D11-Tat antibody could greatly reduce intracellular HBx via Fc binding receptor TRIM21-mediated protein degradation. This process simultaneously stimulated the activations of NF-κB, AP-1, and IFN-ß, which promoted an antiviral state of the host cell. Conclusion: In summary, our study offers a new approach to target intracellular pathogenesis-related protein by engineered cell-penetrating mAb expanding their potential for therapeutic applications. Moreover, the 9D11-Tat antibody may provide a novel therapeutic agent against human chronic HBV infection.

18.
Hepatol Res ; 48(3): E133-E145, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28707778

RESUMO

AIM: Chronic hepatitis B (CHB) patients with normal alanine aminotransferase (ALT) levels are not free from significant hepatic lesions. Recently, there has been an improved understanding of the clinical significance of quantitative hepatitis B core antibody levels (qAnti-HBc) during CHB management. In this cross-sectional study, we evaluated the utility of qAnti-HBc in identifying significant liver inflammation in CHB patients. METHODS: A total of 469 patients (training set, n = 363; validation set, n = 106) who underwent liver biopsy (LB) were included. The qAnti-HBc levels were quantified and the relationship between histology and serum markers was systematically analyzed. RESULTS: In the training set, qAnti-HBc levels were found to have significant diagnostic value for moderate to severe liver inflammation (≥G2) in all patients (area under the receiver operating characteristic curve [AUROC] = 0.768; 95% confidence interval [CI], 0.721-0.810; P < 0.001) and in patients with normal or near-normal ALT levels (AUROC = 0.767; 95% CI, 0.697-0.828; P < 0.001). Our novel index (AC index) for the identification of ≥G2 inflammation, which combined the qAnti-HBc and ALT levels, significantly improved diagnostic performance (AUROC = 0.813; 95% CI, 0.768-0.852) compared to the use of ALT alone (AUROC = 0.779; 95% CI, 0.732-0.821) in all patients. In the validation set, the AC index showed an improved AUROC of 0.890 (95% CI, 0.814-0.942) and 0.867 (95% CI, 0.749-0.943) in all patients and patients with normal ALT levels, respectively. CONCLUSIONS: The qAnti-HBc level predicts significant liver inflammation well, even in patients with normal or near-normal ALT levels. Compared with the conventional ALT level, the AC index is a more reliable non-invasive biomarker for significant liver inflammation in CHB patients.

19.
Nat Commun ; 8(1): 1877, 2017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29187732

RESUMO

A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Especificidade de Anticorpos , Antivirais/uso terapêutico , Cristalografia por Raios X , Células HEK293 , Humanos , Palivizumab/uso terapêutico , Ligação Proteica , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/uso terapêutico , Ressonância de Plasmônio de Superfície
20.
Appl Microbiol Biotechnol ; 101(23-24): 8585-8594, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29038976

RESUMO

Hepatitis E virus (HEV) is one of the major pathogens that cause acute viral hepatitis. The human (genotypes 1 and 2) and zoonotic (genotypes 3 and 4) groups of HEV present different epidemiology and clinical features. In this study, we developed a classification method for rapidly classifying HEV into human or zoonotic groups that combines a general antigen test with a zoonotic group-specific antigen test. Evaluation of serial samples from HEV-infected rhesus monkeys indicated that HEV antigen-positive samples can be classified using the antigen-based classification method. The antigen-based classification method was evaluated further on 55 genotyped samples from acute hepatitis E patients, including 9 human and 46 zoonotic groups. The novel method was completely consistent with the sequencing results: 9/9 for the human groups (100%, 95% confidence interval [CI] 66.4-100%) and 46/46 for the zoonotic groups (100%, 95% CI 92.3-100%). This method was also successfully used for the clustering of some samples that could not be clustered by sequencing. Compared with the sequencing-based method, this method is less time-consuming, less expensive, and less technically complex and is therefore ideal for large numbers of samples. In conclusion, this study provides a convenient and sensitive method for classifying different groups of HEV, and it has potentially important public health applications, especially in underdeveloped areas that cannot afford the high cost of nucleic acid testing.


Assuntos
Antígenos Virais/imunologia , Vírus da Hepatite E/classificação , Vírus da Hepatite E/imunologia , Hepatite E/virologia , Sorotipagem/métodos , Animais , Hepatite E/veterinária , Humanos , Macaca mulatta , Fatores de Tempo
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