Heqiu Zhang

A novel combined vaccine candidate containing epitopes of HCV NS3, core and E1 proteins induces multi-specific immune responses in BALB/c mice

Hepatitis C virus (HCV) has emerged as the major pathogen of liver disease worldwide. The mechanisms of HCV infection and interaction with a host are poorly understood. What exactly is required for efficient control of HCV infection is largely unknown. Standard treatment combining interferon-alpha (IFN-alpha) and ribavirine is effective in about 50% of the treated patients, however associated with significant toxicity and cost. Therefore, the development of new drugs or vaccines is urgently needed. An efficient vaccine against HCV infection requires induction of broad cellular and humoral immune responses against several viral proteins. We have engineered the combined vaccine candidate mT+mE1, an inclusion of multiple epitopes from HCV NS3, core (C) and E1 proteins. mT contains multiple conserved CD4(+) and CD8(+) T cell epitopes from HCV NS3 and C proteins. mE1 is based on eight dominant neutralizing epitopes of E1 protein from six HCV genotypes. In current study, we showed that immunization with mT+mE1 induced high titers of IgG, IgG1 and IgG2a antibodies to mE1, and high level of NS3- or C-specific CTLs. Furthermore, mT+mE1 elicited a Th1-biased immune response with secretion of high amounts of IFN-gamma, compared with mT alone. Prophylactic as well as therapeutic administration of mT+mE1 in BALB/c mice led to protecting mice against SP2/0 tumor cells expressing HCV NS3 protein. These results suggested that mT+mE1 elicited strong humoral immune responses and multiple specific cellular immune responses. The vaccine candidate is now being tested in pre-clinical trials.

Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies.

A novel and simple preparative method for uniform-sized PLGA microspheres: Preliminary application in antitubercular drug delivery.

Particle size has been demonstrated as a key parameter influencing the phagocytosis of drug-loaded PLGA microspheres (MS) by the target cells. However, the current preparative methods were either insufficient in controlling the homogeneity of the produced MS, or requires sophisticated and costly equipment. This study aimed to explore a simple and economical method for uniform PLGA MS preparation. Based on the heterogeneous emulsification of routine mechanical stirring, we designed an adjuvant strategy to enhance the homogeneity of MS. By using glass beads as adjutant, the dispersion produced during mechanical stirring was much more homogeneous in the solution. The particles produced were much smaller and the size distribution was much narrower as compared with those produced using the routine mechanical stirring method under the same condition. After enrichment by selective centrifugation, about 60% of the particles of similar size were obtained, providing further evidence for the efficiency of the novel method in controlling particle homogeneity. Further, the method was applied to prepare rifampicin-loaded PLGA MS of the optimized size for macrophage uptake. The functional evaluation showed that the prepared PLGA MS could efficiently deliver an antitubercular drug into macrophages and maintain a higher intracellular concentration by controlled release, suggesting the potential application of the method in PLGA MS-based drug delivery. Collectively, the study provided a simple and economical method for preparing uniform-sized PLGA MS with potential of widespread applications.

Hepatitis C virus core protein promotes the migration and invasion of hepatocyte via activating transcription of extracellular matrix metalloproteinase inducer

The chronic infection of hepatitis C virus (HCV) becomes a main factor evoking hepatocellular carcinoma, where the HCV core protein plays a central role in hepatocarcinogenesis. Whether the core protein directly contributes to metastasis of hepatocytes still remains to be reported in literature. Transwell chamber migration assay, Boyden chamber invasion assays and scanning electron microscopy observations were performed to determine the prometastatic ability of HCV core protein when expressed in human hepatocyte L02 cells. In addition, western blots, dual-luciferase assays, and chromatin immunoprecipitation assays were used to elucidate HCV core protein dependent pathways that promote metastasis in hepatocytes. Our investigation suggests that HCV core protein markedly enhances the capability of migration and invasion in L02 clones expressing HCV core proteins. The metastasis-promoting effect of the core protein is, in part, highly dependent on its effect on promoting the binding of transcription factor Sp1 to the extracellular matrix metalloproteinase inducer promoter. The effect of Sp1 binding resulted in an increase in extracellular matrix metalloproteinase inducer expression and progression of metastasis. Thus, we report that the expression of HCV core protein contributes to the metastasis of hepatocyte cells through activating transcription of extracellular metalloproteinase inducer.

Non-invasive detection of hepatocellular carcinoma serum metabolic profile through surface-enhanced Raman spectroscopy

The present study aims to identify distinctive Raman spectrum metabolic peaks to predict hepatocellular carcinoma (HCC). We performed a label-free, non-invasive surface-enhanced Raman spectroscopy (SERS) test on 230 serum samples including 47 HCC, 60 normal controls (NC), 68 breast cancer (BC) and 55 lung cancer (LC) by mixing Au@AgNRs with serum directly. Based on the observed SERS spectra, discriminative metabolites including tryptophan, phenylalanine, and etc. were found in HCC, when compared with BC, LC, and NC (P<0.05 in all). Common metabolites-proline, valine, adenine and thymine were found in HCC, BC and LC with compared to NC group (P<0.05). Importantly, Raman spectra of HCC serum biomarker AFP were firstly detected to analyze the HCC prominent peak. Orthogonal partial least squares discriminant analysis was adopted to assess the diagnostic accuracy; area under curve value of HCC is 0.991. This study provides new insights into the HCC metabolites detection through Raman spectroscopy.

Identification of Novel RD1 Antigens and Their Combinations for Diagnosis of Sputum Smear−/Culture+ TB Patients

Rapid and accurate diagnosis of pulmonary tuberculosis (PTB) is an unresolved problem worldwide, especially for sputum smear− (S−) cases. In this study, five antigen genes including Rv3871, Rv3874, Rv3875, Rv3876, and Rv3879 were cloned from Mycobacterium tuberculosis (Mtb) RD1 and overexpressed to generate antigen fragments. These antigens and their combinations were investigated for PTB serodiagnosis. 298 serum samples were collected from active PTB patients, including 117 sputum smear+ (S+) and sputum culture+ (C+) cases, 101 S−/C+ cases, and 80 S−/C− cases. The serum IgG levels of the five antigens were measured by ELISA. Based on IgG levels, the sensitivity/specificity of Rv3871, Rv3874, Rv3875, Rv3876, and Rv3879 for PTB detection was 81.21%/74.74%, 63.09%/94.78%, 32.21%/87.37%, 62.42%/85.26%, and 83.56%/83.16%, respectively. Furthermore, the optimal result for PTB diagnosis was achieved by combining antigens Rv3871, Rv3876, and Rv3879. In addition, the IgG levels of Rv3871, Rv3876, and Rv3879 were found to be higher in S−/C+ PTB patients than in other PTB populations. More importantly, combination of the three antigens demonstrated superior diagnostic performance for both S−/C+ and S−/C− PTB. In conclusion, the combination of Rv3871, Rv3876, and Rv3879 induced higher IgG response in sputum S−/C+ PTB patients and represents a promising biomarker combination for diagnosing of PTB.

Cancerogenic effect of different fragments of the hepatitis C virus core protein.

The hepatitis C virus core protein plays an extremely important role in the hepatocarcinogenesis of hepatitis C virus. Little, however, is known about the oncogenic potency of fragments. Thus, the purpose of the present study is to investigate the cancerogenic effects of the different core protein fragments. Two series of recombinant plasmids containing hepatitis C virus core gene fragments encoding the different-length core protein were constructed using plasmid enhanced green fluorescent protein (pEGFP)-C1 and pcDNA3.1(+), respectively. Human hepatocyte L02 cells transiently transfected with pEGFP-C1-based plasmids were subjected to confocal laser scanning microscopy analysis to determine the localization of the different core protein fragments. The stably transfected L02 cells with the pcDNA3.1(+)-based core protein plasmids were used to investigate the ultrastructural effects of the core protein and the tumorigenicity of L02 cells expressing core protein fragments in athymic nude mice. The full-length core protein and Core130−191 were completely localized in the cytoplasm, while Core1−59 existed exclusively in the nucleus. On the other hand, Core50−140 and Core1−140 were observed in both the nucleus and the cytoplasm. Ultrastructural changes of L02 cells expressing the full-length core protein were comprehensive and included, for example, irregular nuclear, increased nuclear/cytoplasmic ratio and mitochondria swelling. The slight changes were observed in the cells expressing Core50−140 and Core130−191, whereas the ultrastructure of the cells expressing Core1−59 remained normal. All the L02 cells stably expressing different fragments of the core protein, with the exception of the C-terminal truncated fragment Core1−59, could induce the occurrence of tumor in the nude mice. The N-terminal fragment of the core protein, Core1−59, was not oncogenic, while the intermediate and posterior segments of the hepatitis C virus core protein had the cancerogenic potency. In view of the existence of many important immunogenic epitopes in it, the core protein anterior segment might be a safer candidate for the development of hepatitis C virus vaccine.

Construction, Expression, Purification and Biotin Labeling of a Single Recombinant Multi-Epitope Antigen for Double-Antigen Sandwich ELISA to Detect Hepatitis C Virus Antibody

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Construction of an immunostimulatory plasmid, pUCpGs10, and research on its immune adjuvant effect.

In order to overcome the instability of CpG ODN in vivo, sequence diversity, and individual differences, eleven CpG ODN fragments were meticulously selected and linked to form a Multi-CpG, which were repeatedly inserted into the cloning vector pUC19 for constructing the recombinant plasmid pUCpGs10 containing ten of Multi-CpG. Using the multi-genotype HCV E1 and multi-epitope complex HCV-T as immunogens, and plasmid pUCpGs10 as the immune adjuvant, Balb/c mice were immunized through nasal and subcutaneous immunization. Strong-specific humoral and cellular immune response were induced, which can obviously inhibit the growth of homograft expressing HCV antigen. The immune adjuvant effect of pUCpGs10 closely matched that of Freund’s complete adjuvant. The plasmid pUCpGs10 can significantly improve IgA content in serum and different mucosal extract and systematical T-cell response via intranasal immunization. In conclusions, the newly constructed immunostimulatory plasmid pUCpGs10 is able to effectively activate the humoral and cellular immune activity, and possesses activation on mucosal immune response.