Characterization of two excipient interaction degradation products in oseltamivir phosphate powder for oral suspension by MS and NMR.

Oseltamivir phosphate is widely used to treat and prevent influenza, and is available in the form of capsules, powder for oral suspension, pediatric solutions, and granules. Because of the amino group, oseltamivir is easy to react with the excipients of the formulation to generate drug-excipient interaction impurities. In this research, two degradation products in a commercial oseltamivir phosphate powder for oral suspension due to interaction between API and citrate were investigated. They were characterized to be 3-((-6-acetamido-3-(ethoxycarbonyl)-5-(pentan-3-yloxy)cyclohex-3-en-1-yl)carbamoyl)-3-hydroxypentanedioic acid and 2-(2-((-6-acetamido-3-(ethoxycarbonyl)-5-(pentan-3-yloxy)cyclohex-3-en-1-yl)amino)-2-oxoethyl)-2-hydroxysuccinic acid by MS and NMR, respectively. Furthermore, the formation mechanisms of these impurities were verified, and the method of analysis of covariance was used to assess the rate of impurities' degradation. HIGHLIGHTS: Two excipient interaction degradation products in commercial oseltamivir phosphate powder for oral suspension were studied and elucidated in detail via LC-MS/MS and NMR. The incompatibility risk of pH conditioners such as citrate and citric acid with formulations that contain an amino group was disclosed in this article. Analysis of covariance was demonstrated to assess the impact of various formulations and preparation techniques on the rate of impurity degradation.

Effects of hypertension on the outcomes of COVID-19: a multicentre retrospective cohort study.

Objectives: Hypertension is thought to be a contributor to mortality in coronavirus disease 2019 patients; however, limited clinical data on the outcomes of COVID-19 in patients with hypertension are available.Methods: This study was designed to confirm whether hypertension affects the outcomes of COVID-19. Results: A total of 983 patients with COVID-19 (female, 48%; male, 52%) were enrolled. Significantly higher odds of 60-day mortality (p = .017) were observed in the hypertensive group. In the hypertensive group, even after adjustment in multivariate analysis, the subgroup of patients 70 years old and older had higher 28-day mortality and total 60-day mortality rates than the other age subgroups (bothp < .05). A total of 297 (89%) COVID-19 patients with hypertension survived, and 35 (11%) died. In addition, compared with hypertensive patients who survived COVID-19, non-survivors had more pre-existing conditions, including cardiovascular diseases and stroke, higher blood pressure on admission, more severe inflammation, and more liver and kidney damage.Conclusion: Hypertension does not affect the outcome of COVID-19, which is different than the conclusions drawn in other studies. However, the 28-day mortality and total 60-day mortality rates of hypertensive patients (age ≥ 70) with COVID-19 were significantly elevated, and compared with the group of survivors, non-surviving COVID-19 patients with hypertension were older, had more basic diseases and had a more severe clinical condition.

Risk factors for mortality in critically ill patients with COVID-19: a multicenter retrospective case-control study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has spread around the world, until now, the number of positive and death cases is still increasing. Therefore, it remains important to identify risk factors for death in critically patients. METHODS: We collected demographic and clinical data on all severe inpatients with COVID-19. We used univariable and multivariable Cox regression methods to determine the independent risk factors related to likelihood of 28-day and 60-day survival, performing survival curve analysis. RESULTS: Of 325 patients enrolled in the study, Multi-factor Cox analysis showed increasing odds of in-hospital death associated with basic illness (hazard ratio [HR] 6.455, 95% Confidence Interval [CI] 1.658-25.139, P = 0.007), lymphopenia (HR 0.373, 95% CI 0.148-0.944, P = 0.037), higher Sequential Organ Failure Assessment (SOFA) score on admission (HR 1.171, 95% CI 1.013-1.354, P = 0.033) and being critically ill (HR 0.191, 95% CI 0.053-0.687, P = 0.011). Increasing 28-day and 60-day mortality, declining survival time and more serious inflammation and organ failure were associated with lymphocyte count < 0.8 × 109/L, SOFA score > 3, Acute Physiology and Chronic Health Evaluation II (APACHE II) score > 7, PaO2/FiO2 < 200 mmHg, IL-6 > 120 pg/ml, and CRP > 52 mg/L. CONCLUSIONS: Being critically ill and lymphocyte count, SOFA score, APACHE II score, PaO2/FiO2, IL-6, and CRP on admission were associated with poor prognosis in COVID-19 patients.

[Research progress of coagulation dysfunction in coronavirus disease 2019].

The coronavirus disease 2019 (COVID-19) has outbroken globally. As an acute infectious disease, COVID-19 has significant impacts on multiple organs and systems throughout the body. Among patients with COVID-19, especially severe and critical cases, a variety of potential risk factors for coagulation dysfunction exist. Furthermore, the coagulation dysfunction of COVID-19 patients was mainly characterized by elevated D-dimer levels. The coagulation dysfunction could directly affect the prognosis of COVID-19 patients and is a major cause of death in patients with severe COVID-19. In this article, the literatures on the basic clinical manifestations, clinical risk factor, mechanism of coagulation dysfunction and evaluation of coagulation function in COVID-19 were reviewed.

Clinical efficacy of intravenous immunoglobulin therapy in critical ill patients with COVID-19: a multicenter retrospective cohort study.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) outbreak is a major challenge all over the world, without acknowledged treatment. Intravenous immunoglobulin (IVIG) has been recommended to treat critical coronavirus disease 2019 (COVID-19) patients in a few reviews, but the clinical study evidence on its efficacy in COVID-19 patients was lacking. METHODS: 325 patients with laboratory-confirmed critical COVID-19 were enrolled from 4 government-designated COVID-19 treatment centres in southern China from December 2019 to March 2020. The primary outcomes were 28- and 60-day mortality, and the secondary outcomes were the total length of in-hospital and the total duration of the disease. Subgroup analysis was carried out according to clinical classification of COVID-19, IVIG dosage and timing. RESULTS: In the enrolled 325 patients, 174 cases used IVIG and 151 cases did not. The 28-day mortality was improved with IVIG after adjusting confounding in overall cohort (P = 0.0014), and the in-hospital and the total duration of disease were longer in the IVIG group (P < 0.001). Subgroup analysis showed that only in patients with critical type, IVIG could significantly reduce the 28-day mortality, decrease the inflammatory response and improve some organ functions (all P < 0.05); the application of IVIG in the early stage (admission ≤ 7 days) with a high dose (> 15 g per day) exhibited significant reduction in 60-day mortality in the critical-type patients. CONCLUSION: Early administration of IVIG with high dose improves the prognosis of critical-type patients with COVID-19. This study provides important information on clinical application of IVIG in the treatment of SARS-CoV-2 infection, including patient selection and administration dosage and timing.

Thymosin α1 therapy in critically ill patients with COVID-19: A multicenter retrospective cohort study.

BACKGROUND: COVID-19 characterized by refractory hypoxemia increases patient mortality because of immunosuppression effects. This study aimed to evaluate the efficacy of immunomodulatory with thymosin α1 for critical COVID-19 patients. METHODS: This multicenter retrospective cohort study was performed in 8 government-designated treatment centers for COVID-19 patients in China from Dec. 2019 to Mar. 2020. Thymosin α1 was administrated with 1.6 mg qd or q12 h for >5 days. The primary outcomes were the 28-day and 60-day mortality, the secondary outcomes were hospital length of stay and the total duration of the disease. Subgroup analysis was carried out according to clinical classification. RESULTS: Of the 334 enrolled COVID-19 patients, 42 (12.6%) died within 28 days, and 55 (16.5%) died within 60 days of hospitalization. There was a significant difference in the 28-day mortality between the thymosin α1 and non-thymosin α1-treated groups in adjusted model (P = 0.016), without obvious differences in the 60-day mortality and survival time in the overall cohort (P > 0.05). In the subgroup analysis, it was found that thymosin α1 therapy significantly reduced 28-day mortality (Hazards Ratios HR, 0.11, 95% confidence interval CI 0.02-0.63, P=0.013) via improvement of Pa02/FiO2 (P = 0.036) and prolonged the hospital length of stay (P = 0.024) as well as the total duration of the disease (P=0.001) in the critical type patients, especially those aged over 64 years, with white blood cell >6.8×109/L, neutrophil >5.3×109/L, lymphocyte < 0.73 × 109/L, PaO2/FiO2 < 196, SOFA > 3, and acute physiology and chronic health evaluation (APACHE) II > 7. CONCLUSION: These results suggest that treatment with thymosin α1 can markedly decrease 28-day mortality and attenuate acute lung injury in critical type COVID-19 patients.

[Hepatitis B virus X promotes HepG2 cell cycle progression and growth via downregulation expression of p16 protein].

OBJECTIVE: To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma. METHODS: A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting. RESULTS: The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR. CONCLUSION: HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.

[The comparative study on the regulation of apoptosis by hepatitis B virus X protein between B and C genotype].

OBJECTIVE: To investigate the apoptosis regulation on hepatoma cells by HBx between genotype B and C. METHODS: Genotype B and C HBx gene fragments were amplified and inserted into green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP-XB and pGFP-XC. The pEGFP-C1, pGFP-XB and pGFP-XC were introduced into Bel-7402 cells by Fugene HD to obtain Bel-7402 cells expressing GFP. The transcription and expression of HBx gene were demonstrated by RT-PCR and Western Blot analysis. Bel-7402, Bel-7402/GFP, Bel-7402/GFP-XB, Bel-7402/GFP-XC cells were treated with adriamycin (2.5 microg/ml), and the apoptosis of the cells was determined by trypan blue exclusion, and flow cytometry analysis. RESULTS: RT-PCR and Western Blot analysis showed that HBx genes of genotypes B and C were transcribed and expressed in Bel-7402/GFP-XB, Bel-7402/GFP-XC cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in Bel-7402, Bel-7402/GFP cells while no significant cell death was observed in Bel-7402/GFP-XB, Bel-7402/GFP-XC cells. Flow cytometry analysis indicated that no significant differences of apoptosis rates of Bel-7402/GFP-XB (3.87%) and of Bel7402/GFP-XC (4.01%) were observed (P > 0.05), moreover, no significant differences of Bel-7402/ GFP-XB (3.87%), Be17402/GFP-XC (4.01%) and of the untreated cells. Apoptosis rates in Bel-7402/GFP-XB (3.87%), Bel-7402/GFP-XC (4.01%) cells were significantly lower than those in Bel-7402 (27.05%) and Bel-7402/GFP (29.14%) cells at 48 hours after the adriamycin treatment (P < 0.01). CONCLUSIONS: Bel-7402 cell lines expressing GFP, GFP-XB and GFP-XC fusion proteins were successfully established. HBV X protein blocks adriamycin-induced apoptosis of Bel-7402 cells. There is no difference between HBx of genotype B and C in inhibiting apoptosis induced by adriamycin.

[Small interfering RNA targeting to hepatitis B virus X gene and 5-aza-2'-deoxycytidineon inhibited growth of the subcutaneous implanted tumor of hepatocellular carcinoma in nude mice].

OBJECTIVE: To investigate the anti-tumor effect of small interfering RNA targeting to HBV X gene (X-siRNA) and 5-aza-2'-deoxycytidine (5-aza-dC) on HBV-related hepatocellular carcinoma. METHODS: X-siRNA and control siRNA were synthesized. HepG2/GFP-HBx cells were treated with X-siRNA, and the levels of HBV X mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Nude mice were inoculated with HepG2/GFP and HepG2/GFP-HBx cells subcutaneous respectively to establish implant models of hepatocellular carcinoma, and were treated with X-siRNA, 5-aza-dC alone or in combination, and tumor growth was observed. The methylation of p16 gene promoter was detected by methylation specific polymerase chain reaction (MSP). RESULTS: RT-PCR showed the expression of HBV X mRNA in HepG2/GFP-HBx cells was inhibited markedly by X-siRNA. The nude mice experiment showed that the gross tumor volume was much bigger in HepG2/GFP-HBx group than that in HepG2/GFP group (P < 0.05). The growth of palpable tumors in X-siRNA or 5-aza-dC treatment group notably decreased (P < 0.05). MSP analysis showed that p16 gene methylation was observed in HepG2/ GFP-HBx-caused palpable tumors, while no methylation was detected in HepG2/GFP group. However, after treatment with X-siRNA or 5-aza-dC, p16 gene methylation reduced. CONCLUSIONS: HBV X-siRNA and methylation inhibitor can inhibit the growth of hepatoma cells via reversing p16 methylation.

[Efficacy of the 96-week adefovir dipivoxil therapy in patients with chronic hepatitis B].

OBJECTIVE: To investigate the efficacy of the 96-week antiviral therapy with adefovir dipivoxil in patients with chronic hepatitis B. METHODS: 80 patients with chronic hepatitis B received the antiviral therapy of adefovir dipivoxil (ADV, 10 mg/d). At the 12th week, 19 cases without early viral response (EVR, HBV DNA drop < 2 log10copies/ml) switched to the therapy of other nucleoside analogues. Aminotransferase (ALT) normalization, HBV DNA negative, HBeAg loss and HBeAg seroconvertion were accessed at the 96th week. RESULTS: At week 96, ALT normalization and HBV DNA negative in 61 patients with ADV therapy were 85.25% (52/61) and 95.08% (58/61); and HBeAg loss and HBeAg seroconvertion were 52.52% (17/33) and 42.42% (14/33) respectively. While for the other 19 patients switching to other nucleoside analogues, ALT normalization and HBV DNA negative came to 57.89% (11/19) and 68.42% (13/19). Both HBeAg loss and HBeAg seroconvertion were 58.33% (7/12). CONCLUSION: Long term ADV antiviral therapy is effective to inhibit HBV DNA replications and benefits patients with chronic hepatits B. Switching to another nucleoside analogue is an optimal alternative if there is no EVR at week 12 in ADV therapy.

[Hepatitis B virus X protein suppresses adriamycin-induced apoptosis of hepatocellular carcinoma cells and expression of p53 and PTEN].

OBJECTIVE: To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells and the expressions of p53 and PTEN. METHODS: HepG2, HepG2/GFP, and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and the apoptotic cell death was determined by observing the morphological changes and flow cytometry. The expressions of p53 and PTEN mRNA in the 3 cells were detected by RT-PCR, and the expressions of p53 and PTEN protein were analyzed by Western blotting. RESULTS: Adriamycin induced significant cell death in HepG2 and HepG2/GFP cells, which became rounded, shrunk, and detached after the treatment; but no significant cell death occurred in HepG2/GFP-HBx cells. Flow cytometry analysis showed that the apoptotic rate was significantly lower in HepG2/GFP-HBx cells (3.94%) than in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 h after the treatment (P<0.001), while no significant difference was observed between HepG2/GFP-HBx (3.94%) and the control cells (2.12%, 2.78%, and 2.55%) (P>0.05). RT-PCR showed lowered expression of PTEN mRNA in HepG2/GFP-HBx cells as compared to that in HepG2 and HepG2/GFP cells, while no significant difference was noted in p53 mRNA. Western blot analysis showed that PTEN protein decreased while p53 protein remain unchanged in HepG2/GFP-HBx cells. CONCLUSION: HBx suppresses adriamycin-induced apoptosis of HepG2 cells and PTEN expression. The inhibitory effect of HBx on the cell apoptosis may be related to the inhibition of p53-PTEN pathway.

[The antiviral treatment impacts on clinical outcomes of renal transplantation recipients with hepatitis B virus infection].

UNLABELLED: OBJECTIVE; To investigate the clinic outcomes and the efficacy of antiviral treatment in renal transplantation recipients with hepatitis B viral serum markers positive. METHODS: 32 renal transplantation recipients with hepatitis B viral serum markers positive were enrolled. 23 patients in antiviral treatment group have received the lamivudine (19 cases), enticavir (2 cases) and adefovir (1 case). Another 9 patients have not received the antiviral treatment and were as the control group. RESULTS: The biochemical response rate in antiviral treatment group and control group is 82.60% and 22.22%, respectively. 19 of 23 (82.60%) patients in treatment group survived and 1 of 9 (11.11%) patients in control group survived (P < 0.05). 20 of 23 (86.95%) patients in treatment group have the reduction of HBV DNA more than 2 log copies/ml or maintain less than 5 log copies/ml, while 1 of 9 (11.11%) patients in control group has the HBV DNA maintain less than 5 log copies/ml (P < 0.05). The virology rebound was observed in 6 of 19 (31.58%) patients with lamivudine treatment. 2 of them shift to enticavir treatment and 1 of them add adefovir treatment. The three patients survived. Other 3 patients die of liver function failure. CONCLUSION: The antiviral could improve the survival in renal transplantation recipients with hepatitis B viral serum markers positive. When the virology rebound occurs, the add-on with adefovir or the shift to enticavir could be a rescue treatment.

[The relationship between the levels of HBV DNA loads and both the clinical characteristics and 48-week prognosis in patients with decompensated cirrhosis due to hepatitis B].

OBJECTIVE: To evaluate the relationship between the levels of HBV DNA loads and both the clinical characteristics and 48-week prognosis in patients with decompensated cirrhosis due to hepatitis B. METHODS: One hundred and forty-three patients with decompensated cirrhosis of hepatitis B virus infection were divided into low level HBV DNA group [HBV DNA < 10(5) copies/ml = (46 cases) and high-level HBV-DNA group (HBV DNA > or = 10(5) copies/ml) (97 cases)]. 21 cases in low-level group and 52 cases in high-level group treated with nucleoside analog. RESULTS: There was no significant difference between the two groups on the demography and the baseline in ALT, ALB, TBil, CHE before treatment, while in AST and HBeAg were statistically different. At 48-week, there was no significant difference between the two groups on the liver function. The mortality rate in low-level group was similar to that in high level group. In the low-level HBV DNA patients, hepatocellular carcinoma, spontaneous peritonitis and gastrointestinal hemorrhage were higer than that in the high-level HBV DNA patients. CONCLUSION: In patients with decompensated cirrhosis due to hepatitis B, those who were in low-level HBV DNA had not got better than that in high-level HBV DNA, which indicated that earlier treatment was also needed in low-level HBV DNA patients.

[Hepatitis B virus X protein suppressing adriamycin-induced apoptosis of HepG2 cells].

OBJECTIVES: To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells. METHODS: HBx gene fragment was amplified from subtype adr HBV plasmid by PCR, and inserted into Hind III and Kpn I sites of green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP/HBx. The pEGFP-C1 and pGFP-HBx were introduced into HepG2 cells by Lipofectamine 2000 to obtain HepG2 cells expressing GFP. GFP-HBx fusion protein was selected using G418. The expression of HBx gene was demonstrated by RT-PCR analysis. HepG2, HepG2/GFP and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and apoptosis of the cells was determined by their morphological changes, trypan blue exclusion, and flow cytometry analysis. RESULTS: Under a fluorescence microscope, visible expression of GFP and GFP-HBx fusion proteins were observed in HepG2/GFP and HepG2/GFP-HBx cells, even after growing over 70 generations. RT-PCR analysis showed that HBx gene was expressed in HepG2/GFP-HBx cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in HepG2 and HepG2/GFP cells while no significant cell death was observed in HepG2/GFP-HBx cells. Flow cytometry analysis showed that apoptosis rates in HepG2/GFP-HBx (3.94%) cells were significantly lower than those in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 hours after the adriamycin treatment (P < 0.01). No significant differences of apoptosis rates of HepG2/GFP-HBx (3.94%) and of the untreated cells (2.12%, 2.78%, 2.55%) (P > 0.05) were observed. CONCLUSION: A HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.

Acidification induces Bax translocation to the mitochondria and promotes ultraviolet light-induced apoptosis.

It has been suggested that Bax translocation to the mitochondria is related to apoptosis, and that cytosol acidification contributes to apoptosis events. However, the mechanisms remain obscure. We investigated the effect of acidification on Bax translocation and on ultraviolet (UV) light-induced apoptosis. The Bax translocation assay in vitro showed that Bax translocated to the mitochondria at pH 6.5, whereas no Bax translocation was observed at pH 7.4. VHDBB cells expressing the GFP-Bax fusion protein were treated for 12 h with a pH 6.5 DMEM medium, nigericin (5 mug/ml) and UV light (50 J/cm(2)), separately or in combination, and Bax translocation to the mitochondria was determined by SDS-PAGE and Western blot, and apoptotic cell death was detected by flow cytometry. The results showed that some of the Bax translocated to the mitochondria in the cells treated with the normal medium, nigericin and UV in combination, whereas all of the Bax translocated to the mitochondria in the cells treated with the pH 6.5 medium, nigericin and UV in combination. In VHDBB cells treated for 12 h with nigericin, UV alone, and UV and nigericin in combination, the respective rates of apoptotic cell death were 25.08%, 33.25% and 52.88%. In cells treated with pH 6.5 medium and nigericin, pH 6.5 medium and UV, and pH 6.5 medium, nigericin and UV in combination, the respective rates of apoptotic cell death increased to 37.19%, 41.42% and 89.44%. Our results indicated that acidification induces Bax translocation from the cytosol to the mitochondria, and promotes UV light-mediated apoptosis. This suggests that there is a possibility of improving cancer treatment by combining acidification with irradiation or chemotherapeutic drugs.

Construction of exogenous multiple epitopes of helper T lymphocytes and DNA immunization of its chimeric plasmid with HBV pre-S2/S gene.

AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization. METHODS: Artificial HTL epitope, PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL, MTE5. pcMTE5 and pcHB were generated by cloning MTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3. Four chimeric plasmids were constructed by cloning MTE5 into the region of pre-S2 gene (Bam HI), 5' terminal of S gene (HincII, Xba I) and 3' terminal of S gene (Acc I) of pcHB respectively. BALB/c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott IMx AUSAB test kits. RESULTS: The sequences of MTE5 and the 6 constructs of recombinant plasmids were confirmed to be correct by DNA sequencing. The anti-HBs response of the co-inoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only (136.7+/-69.1 mIU/mL vs 27.6+/-17.3 mIU/mL, P<0.01, t = -6.56). Among the 4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54+/-7.68 mIU/mL), and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5. CONCLUSION: Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV. It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL. The antibody responses were very low using DNA immunization in the study. Thus, the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA II antigens should also be evaluated after these chimeric plasmids are expressed in mammalian cell lines.

Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of alpha determinant region.

AIM: Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice. METHODS: HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD, respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA) and immune dot blotting to evaluate its antigenicity in vitro. The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo. RESULTS: Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the alpha determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal anti-HBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106+/-26.7 vs 1 814.4+/-776.3, P<0.01, t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs 6/10, 4.56+/-3.52 mIU/mL vs 27.60+/-17.3 mIU/mL, P = 0.02, t = 2.7). CONCLUSIONS: HBsAg with deleted second-loop of the alpha determinant still has antigenicity, and can also raise weak anti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine for preventing infections of immune escape mutants of HBV.