Objective To investigate the outcome of trans-scaphoid perilunar dislocation treated with bone anchor and AO/ASIF hollow screw.Methods Eight patients with trans-scaphoid perilunar dislocation were treated by Depuy Mitek bone anchor and AO/ASIF hollow screw.The postoperative follow-up period was 6～24 months(average 11.1±5.3 months).All patients were assessed by the Cooney's grading system.Results The outcomes of 8 patients were execellent in 5 cases,good in 2 cases,general in 1 case.6 cases got pain relief with an increase of range of motor and grip strength at certain drgree.Postoperative roentgenogram displayed congruity among wrist bones,with no fracture disunion,no loose internal fixation and no traumatic arthritis.Conclusion The bone anchor and hollow screw have advantages as simple-manipulated and reliable for treating trans-scaphoid perilunar dislocation.

Objective:To investigate the efficacy of an anterolateral femoral chimeric perforator flap combined with vancomycin-loaded calcium sulfate in the treatment of chronic infection after internal fixation of calf fracture with soft tissue defects.Methods:Retrospectively analyzed were the data of 16 patients with chronic infection combined with extensive soft tissue defects after internal fixation of calf fracture who had been admitted to Department of Orthopedics, Shenzhen Hospital Affiliated to Peking University from September 2008 to November 2020. There were 11 males and 5 females, aged from 16 to 62 years (average, 37 years). Infection sites: the upper tibia in 4 cases, the middle and lower tibia in 10 cases, and the middle fibula in 2 cases. According to the Cierny-Mader classification, all patients were anatomical type III and by the host classification, there were 14 cases of type B and 2 cases of type C type. The areas of soft tissue defects ranged from 6 cm × 4 cm to 23 cm × 14 cm. All patients were treated by transplantation of an anterolateral thigh chimeric perforator flap combined with vancomycin-loaded calcium sulfate therapy. At the last follow-up, the curative efficacy was evaluated according to the Paley fracture union scoring.Results:All patients were followed up for 8 to 24 months (mean, 16 months). Complete flap survival was achieved in 15 flaps and partial survival in one. According to the Paley fracture union scoring at the last follow-up, the curative efficacy was evaluated as excellent in 15 cases and as good in one. Both the grafted artificial bone and the tibia and fibula achieved bone union after 6 to 12 months (mean, 8.9 months). Infection with chronic sinus tract pus recurred in one case at post-operative one year. After re-debridement, the infection was controlled and the wound healed. The plate internal fixation was replaced by the unilateral or annular external fixator in 14 patients and retained in 2 patients. The lengths of the bone defects averaged 2.4 cm and the time for the external fixation 10.5 months (from 8 to 14 months).Conclusion:In the treatment of chronic infection after internal fixation of calf fracture with extensive soft tissue defects, the efficacy of an anterolateral femoral chimeric perforator flap combined with vancomycin-loaded calcium sulfate is satisfactory, because the flap can fully cover the bone and soft tissue defects while the vancomycin-loaded calcium sulfate can effectively control the infection.

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

Objective:To compare the optimal conditions, virus yield, viral titer and cell metabolism between culturing influenza virus H1N1 vaccine strain in MDCK and MDCK-G1 cells.Methods:The optimal culture conditions were investigated using chessboard method. The hemagglutination titer, half of the tissue infection dose (TCID 50) and the metabolism of glucose and lactic acid were monitored and compared between the two cell lines. Results:After MDCK-G1 cells were inoculated with H1N1 at the multiplicity of infection (MOI) of 0.001 with the presence of 1 μg/ml of trypsin, the hemagglutination titer reached the peak of 1∶512 at 72 h and the viral titer was 10 7.4TCID 50/ml. In the MDCK cell line group, the hemagglutination titer reached the peak of 1∶256 at 72 h and the viral titer was 10 6.6TCID 50/ml when using H1N1 at MOI=0.0001 and 1 μg/ml of trypsin. Conclusions:MDCK-G1 cells were more suitable than MDCK cells for the proliferation of influenza virus. This study provided reference data for further research on cell-derived influenza vaccine.

Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.

Influenza is a worldwide infectious disease caused by influenza virus. It has posed great challenges on public health and social stability since 1918. At present, vaccination is the most effective way to prevent and control influenza epidemics. Broad-spectrum antiviral drugs and neutralizing antibodies against influenza virus have been widely studied in recent years. Hemagglutinin (HA), which is on the surface of influenza virus, plays an important role in the stage of viral invasion into host cells. It is the main effective antigenic component of current influenza vaccines, as well as the main target of broad-spectrum neutralizing antibodies and broad-spectrum antiviral drugs. This review summarized the progress in the development of novel influenza vaccines, neutralizing antibodies, and antiviral drugs based on influenza virus HA, as well as other prevention and control measures, hoping to present new ideas for future influenza prevention and control.

Objective: To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus. Methods: Core 700 was firstly used to remove residual host cell proteins, and then Capto Q was used to remove host cell DNA. Several batches of H5N1 influenza A virus cultured in Madin-Darby canine kidney (MDCK) cells were purified using this method. The efficiency of purification was evaluated using many methods including quantitative real-time PCR, hemagglutination (HA) test and single radial immunodiffusion assay. Moreover, Benzonase nuclease was used for comparison. Results: Without the use of Benzonase nuclease, the overall removal rates of host cell DNA and residual proteins were 99.62% and 98.1%, and the HA antigen recovery rate was 66.96%. Conclusions This study established a purification strategy with good effect for cell-based influenza vaccines. It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate. The purification result is no worse than that of adding Benzonase nuclease, suggesting the potential of its application in actual vaccine production.

Objective: To investigate the best amount of TPCK trypsin in Madin Darby canine kidney (MDCK) cell suspension for the culture of H7N9 avian influenza virus. Methods: Different concentrations of TPCK trypsin were added during the periods of cell growth and virus production. Their effects on cell growth, viability, glucose and lactate metabolism, and hemagglutination titer were monitored every 12 h. Inter-batch differences were analyzed. The amount of trypsin added in the cell growth phase was 0, 1 μg/ml, 2 μg/ml, 4 μg/ml, 6 μg/ml, 8 μg/ml, 10 μg/ml and 15 μg/ml. The amount of trypsin added during the virus production period was 0, 0.5 μg/ml, 1 μg/ml, 1.5 μg/ml, 2 μg/ml and 2.5 μg/ml. When the hemagglutination titers were same, the adding amount was further optimized at different multiplicity of infection (MOI) of 0.001, 0.005, 0.025 and 0.05. Results: No significant linear effects of TPCK trypsin concentration on cell number, viability, and glucose and lactate metabolism were observed. No toxicity to cell growth was observed when TPCK trypsin concentration reached 15 μg/ml. After the inoculation of H7N9 avian influenza virus, the hemagglutination titers in the 1 μg/ml, 1.5 μg/ml, 2 μg/ml and 2.5 μg/ml TPCK trypsin groups reached the peaks at 48 h, which were 1∶2^6.5. At 60 h, the hemagglutination titers of the latter two groups decreased faster than those of the former two groups. When the MOI was 0.005, the hemagglutination titer of the 1.5 μg/ml group at 48 h was 2^6.5 higher than 2⁶ in the 1 μg/ml group under the same condition. There were differences between different batches of TPCK trypsin. Conclusions Adding 1 μg/ml and 1.5 μg/ml of trypsin could better promote the proliferation of H7N9 avian influenza virus, and 1.5 μg/ml of trypsin had a wider range of MOI applicability.