Generation and efficacy evaluation of recombinant classical swine fever virus E2 glycoprotein expressed in stable transgenic mammalian cell line.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2) that could stably produce a secreted form of CSFV E2 protein (mE2). The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1:40 to 1:320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1:10,240 to 1:81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1:80 to 1:10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1 × 10(5) TCID50. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3-4 days after challenge and were euthanized from 7-9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine.

Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate.

BACKGROUND: Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis in most Asian regions. There is no specific treatment available for Japanese encephalitis, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. The purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of JEV for potential use of JEV subunit vaccine. RESULTS: We generated a new cell clone (BJ-ME cells) that stably produces a secreted form of Japanese encephalitis virus (JEV) virus-like particle (VLP). The BJ-ME cells were engineered by transfecting BHK-21 cells with a code-optimized cDNA encoding JEV prM and E protein expression plasmid. Cell line BJ-ME can stably produces a secreted form of Japanese encephalitis virus virus-like particle (JEV-VLP) which contains the JEV envelope glycoprotein (E) and membrane protein (M). The amount of JEV-VLP antigen released into the culture fluid of BJ-ME cells was as high as 15-20 µg/ml. JEV-VLP production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. Cell culture fluid containing the JEV-VLP antigen could be harvested five to seven times continuously at intervals of 4-6 days while maintaining the culture. Mice immunized with the JEV-VLP antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. CONCLUSION: These results suggest that the recombinant JEV-VLP antigen produced by the BJ-ME cell line is an effective, safe and affordable subunit Japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse.

Comprehensive mapping of a novel NS1 epitope conserved in flaviviruses within the Japanese encephalitis virus serocomplex.

Nonstructural protein-1 (NS1) of the Japanese encephalitis virus (JEV) is an immunogenic protein that is a potential candidate for the development of vaccines and diagnostic reagents. NS1 is known to be more specific than the E protein in serological testing of flavivirus infections. However, NS1 exhibits cross-reactivity among flaviviruses even within the same genus and more so within a serocomplex. However, the cross-reactive epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of a linear B-cell epitope that is common and specific to the JEV serocomplex of Flaviviridae. We generated an NS1-specific monoclonal antibody that cross-reacts with the West Nile virus (WNV) NS1 protein by immunizing mice with recombinant JEV NS1. For epitope mapping, 51 partially overlapping peptides spanning the entire NS1 protein were expressed with a glutathione S-transferase (GST) tag and screened using monoclonal antibodies. Two linear epitope-containing peptides were identified using enzyme-linked immunosorbent assay (ELISA). By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, we successfully identified the smallest unit of the linear epitope required to react with the monoclonal antibody. The linear epitope was located in amino acids residues ²²7ETHTLW²³². Furthermore, results of the sequence alignment revealed that the epitope was highly conserved among JEV strains. Notably, the epitope is highly conserved among viruses of the JEV serocomplex. Furthermore, the homologous regions on NS1 proteins from dengue viruses showed no cross-reactivity with the monoclonal antibodies. The epitope was recognized by antisera against the WNV but not against the dengue virus. This novel JEV serocomplex-specific linear B-cell epitope of NS1 would be helpful in the development of new vaccines and diagnostic assays.

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5)AIDITRK(11), (72)RDELNVL(78), (251)KSKHNRREGY(260), (269)DENGIVLD(276), and (341)DETTLVRS(348). Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

[The role of BCR/ABL isoforms in the presentations and outcome of Philadelphia-positive acute lymphoblastic leukemia in adult patients].

OBJECTIVE: To investigate the difference of clinical characteristics and outcomes between different isoforms of BCR/ABL in adults with Philadelphia-positive acute lymphoblastic leukemia (ALL). METHODS: The data of 106 adults with Ph+ ALL diagnosed in our hospital from January 1, 1996 to December 31, 2007 were reviewed. The difference of clinical characteristics between different subgroups of BCR/ABL was compared and their relation with outcomes was studied. RESULTS: The median age of the 106 patients was 34 years and the median white blood cell count at baseline was 28.5 x 10(9)/L. Comparative analysis demonstrated that patients in p210 group had an older age, higher blood platelet count (BPC) and more frequent occurrence of splenomegaly. Referring to the outcomes, the complete remission (CR) rate of the two groups were 92.2% and 93.9%, respectively. The median overall survival (OS) and relapse free survival (RFS) in p190 group were 13 months and 10 months, the 1, 3-year estimated OS were (54.7 +/- 6.7)% and (5.5 +/- 5.2)%, and the 1, 3-year estimated RFS were (40.2 +/- 6.8)% and (7.8 +/- 6.7)%, while in p210 group, the median OS and RFS were 15 months and 10 months, respectively, the 1, 3-year estimated OS were (65.8 +/- 8.9)% and (14.5 +/- 7.4)%, and the 1, 3-year estimated RFS were (48.3 +/- 9.4)% and (12.9 +/- 7.7)%. All of the above data had no statistic significance between the two groups. CONCLUSION: Majority of the adults with Ph+ ALL is p190 positive and patients with p210 have older age, higher BPC and more frequent occurrence of splenomegaly, while there is no significant difference between p190 group and p210 group in CR rate, RFS and OS.

[Cytogenetic and prognostic analysis in adult patients with Philadelphia chromosome-positive and bcr-abl positive acute lymphoblastic leukaemia].

OBJECTIVE: To analyze the characteristics of cytogenetic aberration of adults with Philadelphia chromosome-positive (Ph+) and/or bcr-abl positive (bcr-abl+) acute lymphoblastic leukaemia (ALL), and investigate its influence on patients' outcomes. METHOD: Retrospective analysis of 100 adult Ph+ ALL patients from January 1, 1996 to December 31, 2007 was carried out. The type, distribution and frequency of chromosome aberration were summarized, and compared among different subgroups. RESULTS: 1) In all cases, 72 had chromosome aberrations, including 22 with sole Ph chromosome, 44 Ph+ with additional abnormalities, which included double Ph, monosomy 7, monosomy 20, trisomy 8 trisomy 21, 9p deletion and 22 deletion. 2) Patients with pseudodiploid and hyperdiploid had higher WBC count, and inferior outcome with lower rates of overall survival (OS) and relapse free survival (RFS). 3) Ph+ group also had higher WBC counts and inferior outcome with low OS and RFS rates. There was no statistic significance between sole Ph+ group and Ph plus additional aberrations group. 4) Patients with both abnormal and normal metaphase (AN) and with solely abnormal metaphase (AA) had higher WBC count, less frequent P190 occurrence and inferior outcome than those only normal metaphase (NN) group, whereas, there was no difference between AA and AN groups. 5) Double Ph chromosome had a lower frequency of P190 and inferior OS than non-double Ph group. CONCLUSION: Adults with Ph+ ALL have complicated cytogenetic abnormalities, pseudodiploid and hyperdiploid indicate inferior outcome, and double Ph chromosome may be a unfavorable prognostic factor.

[The efficacy of thalidomide for multiple myeloma: a clinical analysis of 102 Chinese patients].

OBJECTIVE: To analyse the efficacy and safety of thalidomide (Thal) for patients with multiple myeloma (MM). METHODS: Effectiveness and adverse events of 102 MM patients treated with thalidomide at a median dosage of 200 mg/d. Thirteen cases were treated with Thal alone (group A), and 105 case with Thal in combination with other therapeutic agents (group B) were retrospectively analyzed. RESULT: 1) The response rate (RR) (CR + PR) was 65.4% for induction therapy in 52 cases and 45.5% for salvage therapy in 66 cases. RR in group B was higher than that in group A (58.1% versus 23.1/%, P= 0.017), and the non-response/progress (NR) rate was lower (15.2% versus 46.2%, P= 0.015). In group B, the NR rate was lower in 50 cases of newly diagnosed MM than in 55 cases of refractory or relapsed MM (6.0% versus 23.6%, P=0.012). In group B, RR between Thal+VAD or M, regimen (72 cases) and Thal + MP regimen (33 cases) was not statistically significant (62.5% versus 48.5%, P >0.05). 2) The median duration of response maintenance was 15.5 (1.0-58.0) months in 21 cases. 3) Among 97 patients with follow-up data, the estimated median duration of OS was 44 months in a median follow-up duration of 20 months and the accumulative time for use of Thal was 8 months. In univariate analysis,the accumulative duration for use of Thal 6 months, hemoglobin > or = 100 g/L and bone marrow megakaryocytes > 20 per smear were associated with longer OS (P = 0.0014, 0.0101, 0.019, respectively). 4) Multivariate analysis suggested that the accumulative time for use of Thal and bone marrow megakaryocytes > 20 were independent good prognostic factors for OS (P = 0.006, 0.036, respectively). 5) The adverse events of Thal were mostly endurable, the rate of thrombus events was lower than that reported in literature. CONCLUSION: Thalidomide alone or combined with chemotherapy is an useful therapy for MM. The accumulative time for use longer than 6 months may improve survival.

[Effect of shRNA targeted to beta-catenin on K562 cell growth].

In order to investigate the effect of shRNA targeted to beta-catenin on the growth of K562 cells, plasmid containing beta-catenin specific shRNA sequence was transfected into K562 cells by lipofectamine 2000, and G418 was added to screen the positive cells. Real-time PCR and Western blot were used to detect the expression of beta-catenin. Cell growth curve, MTT and colony forming cell assays were used to evaluate the proliferation potential of cells. The results showed that the mRNA level of beta-catenin was reduced significantly in K562 cells transfected into interfering plasmid as compared with control plasmid, while the protein level failed to demonstrate difference by the time of 72 hours after transfection. After long-term culture with G418, the count of positive cells enhanced in control group while no positive cells survived in the interfering group. Colony-forming cell assays revealed that the K562 cells in interfering group formed colonies with very small size and low forming rate, compared with the control group, though the growth curve and MTT failed to illustrate differences. It is concluded that the beta-catenin-specific shRNA mediated by plasmid can effectively knockdown the expression of beta-catenin gene and inhibit the colony-forming ability in K562 cells, it is a potential target for the therapy of CML, even in blast crisis.