Affinity maturation to improve human monoclonal antibody neutralization potency and breadth against hepatitis C virus

A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development.     

In vitro production of antibody to hepatitis B core and E antigens by peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

To evaluate the relative immunogenicity of and the mechanism for production of antibody to hepatitis B core (HBc) and hepatitis B e (HBe) Ag, we investigated the in vitro anti-HBc and anti-HBe production by PBMC from 25 patients with chronic active hepatitis (CAH) (15 with HBeAg and 10 with anti-HBe) and 12 ASC (5 with HBeAg and 7 with anti-HBe) in the presence of PWM, rHBcAg, or purified HBeAg. PWM-stimulated culture produced higher titer anti-HBc (mean % inhibition +/- SD = 73 +/- 23%, p less than 0.001) than anti-HBe (34 +/- 17%). HBcAg stimulation elicited greater anti-HBc response (43 +/- 26%, p less than 0.001) than did HBeAg for anti-HBe (26 +/- 12%). Both HBcAg and HBeAg induced equivalent anti-HBe response. Anti-HBc production in response to HBcAg was higher in CAH patients (51 to 55%) than in asymptomatic carriers of hepatitis B surface Ag (22 to 28%) irrespective of their HBeAg/anti-HBe status, but reflecting serum anti-HBc value. Similar findings were noted in HBeAg-stimulated anti-HBe production for the two patient groups. In HBeAg- and anti-HBe-positive CAH, HBcAG-stimulated anti-HBc production was similar in T (1.4 x 10(6)) and B (0.6 x 10(6)) cells coculture, and B cells (2 x 10(6)) alone culture. However, in the HBeAg-stimulated culture, T plus B cells produced significantly higher titer anti-HBe than B cells alone did. These results indicate that HBcAg has a relatively higher immunogenicity in terms of antibody production as compared to HBeAg. Furthermore, HBcAg was shown to function as a T cell-dependent and -independent Ag, whereas HBeAg is T cell-dependent during chronic hepatitis virus B infection in man.     

Expression of hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-HBs antibody

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying a hexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5 % of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100 % with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.     

自然人群乙型肝炎病毒慢性感染者HBV DNA的流行病学分析

目的了解寿光市自然人群乙型肝炎病毒(hepatitis B virus,HBV)慢性感染者HBV DNA的流行病学特征,为乙型肝炎的防治提供依据。方法采集2012年寿光市3个镇街道居民体检发现的HBs Ag阳性者静脉血5 m L,以实时荧光定量聚合酶链反应(FQ-PCR)法检测血清HBV DNA载量,分析HBV DNA与性别、年龄、有无乙肝/肝癌家族史、密切接触史、输血史等因素的相关性。结果在2 026例HBs Ag阳性感染者中,HBV DNA阳性率为49.41%(1 001/2 026),HBV DNA载量的平均值为6.27×107拷贝/m L。其中男性HBV DNA阳性率为53.28%(585/1 098),女性为44.83%(416/928),差异有统计学意义(χ2=14.370,P<0.01);HBV DNA均值男性为(6.72×107±2.07×108)拷贝/m L,女性为(5.56×107±1.44×108)拷贝/m L,差异无统计学意义(t=0.447,P>0.05)。HBVDNA阳性率与年龄呈负相关(r=-0.983),与HBV感染的主要影响因素乙肝/肝癌家族史、密切接触史、输血史等均无关。结论 HBV DNA阳性率与慢性HBV感染者的性别、年龄有关,与HBV感染的主要影响因素无关。     

Prevalence of HIV antigens in cerebrospinal fluid and in serum of patients with both asymptomatic and symptomatic HIV infection]

Prevalence of HIV-Ag in both serum and CSF has been determined in 19 HIV infected patients, including 7 patients without any symptoms or only generalized lymphadenopathy, 5 patients with ARC and 7 patients with AIDS. The results have been correlated with clinically evident neurological disorders. HIV-Ag have been detected in 9 out of 12 patients with ARC (AIDS Related Complex) and AIDS. In 8 of them neurological disorders have been present. Out of the remaining 7 patients in only one HIV-Ag has been detected in CSF (p < 025). No correlation between the presence of HIV antigen in CSF and serum has been noted.     

Cytokine expression during chronic versus occult hepatitis B virus infection in HIV co-infected individuals

Chronic hepatitis B virus infection is characterized by persistent detectable levels of hepatitis B surface antigen (HBsAg) and HBV DNA in the serum. In contrast, HBsAg is not detectable during occult HBV infection, despite the presence of HBV DNA. An altered host immune response could play a role in the development of occult HBV infection; however, potential differences in immune responses among chronic and occult HBV-infected patients have not been evaluated in vivo. In the current study, we evaluated serum levels of regulatory, apoptotic, and fibrotic/anti-fibrotic cytokines/markers as indicators of immune responses in 25 chronic and 12 occult HBV-infected patients. More than half of the patients in both chronic and occult HBV infection groups had IL-2, IL-4, IL-13, and IFN-γ levels below detectable limits. In contrast, most patients had detectable levels of IL-8, IL-10, IP-10, sFas, sFasL, and TGF-β1. Of these, only sFas was significantly different between the two groups, with lower levels observed during occult compared to chronic HBV infection (p = 0.01). As a surrogate marker of apoptotic inhibition, decreased sFas during occult HBV infection suggests that apoptosis occurs at different rates in occult compared to chronic HBV infection and therefore, may contribute to persistence of occult HBV infection.     

Immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) as a marker of interferon therapy in patients with persistent hepatitis B virus infection

Immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) was measured by radioimmunoassay in the sera of 96 HBV carriers. IgM anti-HBc was detected in 17 of 66 patients with chronic active hepatitis and in 4 of 11 with liver cirrhosis. This antibody was not present in asymptomatic carriers or in patients with chronic persistent hepatitis. Testing of sequential samples revealed that the presence of IgM anti-HBc indicated active replication of HBV and at the same time an immune response to the virus. The relationship between IgM anti-HBc and the response to interferon (IFN) therapy was also studied. Results showed that IgM anti-HBc is a useful marker of the efficacy of interferon therapy.     

Detection of a new antibody system reacting with Dane particles in hepatitis B virus infection.

Antibodies in the serum reacting with antigens on the surface of radiolabelled Dane particles distinct from hepatitis B surface and core antigens (HBsAg and HBcAg) were detected, using a double antibody precipitation assay, in 12 out of 15 patients early in the course of acute type B hepatitis and at the time of disappearance of circulating Dane particles. No such antibody activity was found in 15 of the 16 patients with HBsAg-positive chronic active hepatitis, 13 of whom had complete Dane particles in the serum. In a group of 16 asymptomatic HBsAg carriers (without Dane particles in serum) antibody activity was shown in nine. This demonstration of antibodies precipitating Dane particles may be relevant to the clearance of circulating hepatitis B virions and the termination of infection in acute type B hepatitis. Their absence in all but one of the cases of chronic active hepatitis might explain why the virus infection persists in this group of patients.     

Effect of concurrent acute infection with hepatitis C virus on acute hepatitis B virus infection.

OBJECTIVE--To investigate the possible interference with acute hepatitis B virus infection by co-infection with hepatitis C virus. DESIGN--Analysis of stored sera collected for transfusion transmitted viruses study in 1970s. SETTING--Four major medical centres in the United States. PATIENTS--12 recipients of blood infected with hepatitis B virus. MAIN OUTCOME MEASURES--In 1970s, presence of antibodies in hepatitis B virus and raised serum alanine aminotransferase concentration; detection of antibodies to hepatitis C virus with new enzyme linked immunoassays. RESULTS--Five of the 12 patients were coinfected with hepatitis C virus. Hepatitis B surface antigen was first detected at day 59 in patients infected with hepatitis B virus alone and at day 97 in those coinfected with hepatitis C virus (p = 0.01); median durations of antigenaemia were 83 and 21 days respectively (p = 0.05), and the antigen concentration was lower in the coinfected patients. Alanine aminotransferase patterns were uniphasic when hepatitis B virus infection occurred alone (range 479-2465 IU/l) and biphasic in patients with combined acute infection (no value > 380 IU/l; p = 0.0025). Four coinfected recipients developed chronic hepatitis C virus infection. The fifth patient was followed for only four months. CONCLUSIONS--Acute coinfection with hepatitis C virus and hepatitis B virus inhibits hepatitis B virus infection in humans, and onset of hepatitis B may reduce the severity of hepatitis C virus infection but not frequency of chronicity. Alanine aminotransferase concentration showed a biphasic pattern in dual infection.     

2型糖尿病患者乙型肝炎病毒感染率分析

目的：通过调查糖尿病患者乙型肝炎病毒（HBV）的携带率,阐明糖尿病发生是否与HBV感染有关,并观察糖尿病合并HBV感染时是否加重对肝功能的损害。方法：用ELISA方法检测533例2型糖糖尿病患者和1440例普通人群乙型肝炎表面抗原（HBsAg）以及HBV其他血清学标志,同时常规测定肝功能和血糖等。结果：2型糖尿病HBsAg阳性24例,阳性率4.50%,普通人群HBsAg阳性70例,阳性率4.86%,两组间阳性率差异无统计学意义（x2=0.110,P=0.740）。糖尿病患者感染HBV后的抗体应答与普通人群相似。糖尿病合并HBV感染者与无糖尿病HBV感染者的肝功能异常差异无统计学意义。糖尿病患者中83例（16.3%）谷氨酰转肽酶升高,在肝功能异常指标中最常见,这与HBV感染后丙氨酸转氨酶升高为主不同。结论：糖尿病发生与HBV感染无关,糖尿病合并HBV感染时不加重对肝脏的损害。     

Detection of hepatitis B virus in bone allografts from donors with occult hepatitis B infection

The implementation of nucleic acid testing in donor screening has improved the safety of tissue allografts. Although infectious disease transmission can be considered a rare event, the detection of occult hepatitis B infection remains challenging. The studies concerning this risk are mainly based on testing blood specimens. This work shows the correlation between results of samples obtained from donor blood and the corresponding tissue washing solution. Hepatitis B virus deoxyribonucleic acid was detected both in bone allografts from donors with serological profiles associated to active hepatitis B infection and occult hepatitis B infection. These results suggest that hepatitis B virus seems to concentrate in bone marrow even when a low viral load is present in peripheral blood. Even detection at molecular level is not enough to avoid the risk of hepatitis B virus transmission and a multiparametrical evaluation is required in tissue donor screening. The role of clinicians in recognition and reporting of allograft-associated infections is a major concern for the acquisition of experience to be applied in risk control of disease transmission.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.     

Quantitative assay of CD8+ (cytotoxic and suppressor) lymphocytes in patients with both asymptomatic and symptomatic HIV infection]

Lymphocytes CD8+ have been assayed prospectively in 245 individuals infected with HIV. Percentage and number of CD8+ have been nearly two-fold higher in asymptomatic patients or patients with lymphadenopathy than those in the control group. The number of CD8+ lymphocytes has been rapidly decreasing parallel to the progression of HIV (ARC and AIDS), while their percentage has increased--however insignificantly. There has been a positive correlation between the number of CD4+ and CD8+ cells and all clinical stages of HIV infection.