The feasibility of convalescent plasma therapy in severe COVID-19 patients: a pilot study

Currently, there are no approved specific antiviral agents for 2019 novel coronavirus disease (COVID-19). In this study, ten severe patients confirmed by real-time viral RNA test were enrolled prospectively. One dose of 200 mL convalescent plasma (CP) derived from recently recovered donors with the neutralizing antibody titers above 1:640 was transfused to the patients as an addition to maximal supportive care and antiviral agents. The primary endpoint was the safety of CP transfusion. The second endpoints were the improvement of clinical symptoms and laboratory parameters within 3 days after CP transfusion. The median time from onset of illness to CP transfusion was 16.5 days. After CP transfusion, the level of neutralizing antibody increased rapidly up to 1:640 in five cases, while that of the other four cases maintained at a high level (1:640). The clinical symptoms were significantly improved along with increase of oxyhemoglobin saturation within 3 days. Several parameters tended to improve as compared to pre-transfusion, including increased lymphocyte counts (0.65x109/L vs. 0.76x109/L) and decreased C-reactive protein (55.98 mg/L vs. 18.13 mg/L). Radiological examinations showed varying degrees of absorption of lung lesionswithin 7 days. The viral load was undetectable after transfusion in seven patients who had previous viremia. No severe adverse effects were observed. This study showed CP therapy was welltolerated and could potentially improve the clinical outcomes through neutralizing viremia in severe COVID-19 cases. The optimal dose and time point, as well as the clinical benefit of CP therapy, needs further investigation in larger well-controlled trials. Significance StatementCOVID-19 is currently a big threat to global health. However, no specific antiviral agents are available for its treatment. In this work, we explored the feasibility of convalescent plasma (CP) transfusion to rescue severe patients. The results from 10 severe adult cases showed that one dose (200 mL) of CP was welltolerated and could significantly increase or maintain the neutralizing antibodies at a high level, leading to disappearance of viremia in 7 days. Meanwhile, clinical symptoms and paraclinical criteria rapidly improved within 3 days. Radiological examination showed varying degrees of absorption of lung lesions within 7 days. These results indicate that CP can serve as a promising rescue option for severe COVID-19 while the randomized trial is warranted.

Optimization of the amount of TPCK trypsin adding to MDCK cell suspension for culturing H7N9 avian influenza virus / 中华微生物学和免疫学杂志

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∶26.5. 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 26.5 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.

Purification method for cell-cultured influenza virus H5N1 / 中华微生物学和免疫学杂志

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.

Purification method for cell-cultured influenza virus H5N1 / 中华微生物学和免疫学杂志

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 Ma-din-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.