Immunogenicity, safety and immune persistence of the sequential booster with the recombinant protein-based COVID-19 vaccine (CHO cell) in healthy people aged 18-84 years / 中华预防医学杂志

Objective: To evaluate the immunogenicity, safety, and immune persistence of the sequential booster with the recombinant protein-based COVID-19 vaccine (CHO cell) in healthy people aged 18-84 years. Methods: An open-label, multi-center trial was conducted in October 2021. The eligible healthy individuals, aged 18-84 years who had completed primary immunization with the inactivated COVID-19 vaccine 3 to 9 months before, were recruited from Shangyu district of Shaoxing and Kaihua county of Quzhou, Zhejiang province. All participants were divided into three groups based on the differences in prime-boost intervals: Group A (3-4 months), Group B (5-6 months) and Group C (7-9 months), with 320 persons per group. All participants received the recombinant COVID-19 vaccine (CHO cell). Blood samples were collected before the vaccination and after receiving the booster at 14 days, 30 days, and 180 days for analysis of GMTs, antibody positivity rates, and seroconversion rates. All adverse events were collected within one month and serious adverse events were collected within six months. The incidences of adverse reactions were analyzed after the booster. Results: The age of 960 participants was (52.3±11.5) years old, and 47.4% were males (455). The GMTs of Groups B and C were 65.26 (54.51-78.12) and 60.97 (50.61-73.45) at 14 days after the booster, both higher than Group A's 44.79 (36.94-54.30) (P value<0.05). The GMTs of Groups B and C were 23.95 (20.18-28.42) and 27.98 (23.45-33.39) at 30 days after the booster, both higher than Group A's 15.71 (13.24-18.63) (P value <0.05). At 14 days after the booster, the antibody positivity rates in Groups A, B, and C were 91.69% (276/301), 94.38% (302/320), and 93.95% (295/314), respectively. The seroconversion rates in the three groups were 90.37% (272/301), 93.75% (300/320), and 93.31% (293/314), respectively. There was no significant difference among these rates in the three groups (all P values >0.05). At 30 days after the booster, antibody positivity rates in Groups A, B, and C were 79.60% (238/299), 87.74% (279/318), and 90.48% (285/315), respectively. The seroconversion rates in the three groups were 76.92% (230/299), 85.85% (273/318), and 88.25% (278/315), respectively. There was a significant difference among these rates in the three groups (all P values <0.001). During the sequential booster immunization, the incidence of adverse events in 960 participants was 15.31% (147/960), with rates of about 14.38% (46/320), 17.50% (56/320), and 14.06% (45/320) in Groups A, B, and C, respectively. The incidence of adverse reactions was 8.02% (77/960), with rates of about 7.50% (24/320), 6.88% (22/320), and 9.69% (31/320) in Groups A, B, and C, respectively. No serious adverse events related to the booster were reported. Conclusion: Healthy individuals aged 18-84 years, who had completed primary immunization with the inactivated COVID-19 vaccine 3 to 9 months before, have good immunogenicity and safety profiles following the sequential booster with the recombinant COVID-19 vaccine (CHO cell).

Immunogenicity, safety and immune persistence of the sequential booster with the recombinant protein-based COVID-19 vaccine (CHO cell) in healthy people aged 18-84 years / 中华预防医学杂志

Objective: To evaluate the immunogenicity, safety, and immune persistence of the sequential booster with the recombinant protein-based COVID-19 vaccine (CHO cell) in healthy people aged 18-84 years. Methods: An open-label, multi-center trial was conducted in October 2021. The eligible healthy individuals, aged 18-84 years who had completed primary immunization with the inactivated COVID-19 vaccine 3 to 9 months before, were recruited from Shangyu district of Shaoxing and Kaihua county of Quzhou, Zhejiang province. All participants were divided into three groups based on the differences in prime-boost intervals: Group A (3-4 months), Group B (5-6 months) and Group C (7-9 months), with 320 persons per group. All participants received the recombinant COVID-19 vaccine (CHO cell). Blood samples were collected before the vaccination and after receiving the booster at 14 days, 30 days, and 180 days for analysis of GMTs, antibody positivity rates, and seroconversion rates. All adverse events were collected within one month and serious adverse events were collected within six months. The incidences of adverse reactions were analyzed after the booster. Results: The age of 960 participants was (52.3±11.5) years old, and 47.4% were males (455). The GMTs of Groups B and C were 65.26 (54.51-78.12) and 60.97 (50.61-73.45) at 14 days after the booster, both higher than Group A's 44.79 (36.94-54.30) (P value<0.05). The GMTs of Groups B and C were 23.95 (20.18-28.42) and 27.98 (23.45-33.39) at 30 days after the booster, both higher than Group A's 15.71 (13.24-18.63) (P value <0.05). At 14 days after the booster, the antibody positivity rates in Groups A, B, and C were 91.69% (276/301), 94.38% (302/320), and 93.95% (295/314), respectively. The seroconversion rates in the three groups were 90.37% (272/301), 93.75% (300/320), and 93.31% (293/314), respectively. There was no significant difference among these rates in the three groups (all P values >0.05). At 30 days after the booster, antibody positivity rates in Groups A, B, and C were 79.60% (238/299), 87.74% (279/318), and 90.48% (285/315), respectively. The seroconversion rates in the three groups were 76.92% (230/299), 85.85% (273/318), and 88.25% (278/315), respectively. There was a significant difference among these rates in the three groups (all P values <0.001). During the sequential booster immunization, the incidence of adverse events in 960 participants was 15.31% (147/960), with rates of about 14.38% (46/320), 17.50% (56/320), and 14.06% (45/320) in Groups A, B, and C, respectively. The incidence of adverse reactions was 8.02% (77/960), with rates of about 7.50% (24/320), 6.88% (22/320), and 9.69% (31/320) in Groups A, B, and C, respectively. No serious adverse events related to the booster were reported. Conclusion: Healthy individuals aged 18-84 years, who had completed primary immunization with the inactivated COVID-19 vaccine 3 to 9 months before, have good immunogenicity and safety profiles following the sequential booster with the recombinant COVID-19 vaccine (CHO cell).

Advances in epigenetic regulation of Chinese hamster ovary cells / 生物工程学报

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

Optimization and application of caprylic acid precipitation in the purification of monoclonal antibody / 生物工程学报

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.

Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line / 生物工程学报

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Optimization of retinin expression and the application with wax emulsion in nanocoatings / 生物工程学报

Anti-reflective nanocoatings that mimic the eyes of fruit flies are biodegradable materials with great market potential for a variety of optical devices that require anti-reflective properties. Microbial expression of retinin provides a new idea for the preparation of nanocoatings under mild conditions compared to physicochemical methods. However, the current expression level of retinin, the key to anti-reflective coating, is low and difficult to meet mass production. In this study, we analyzed and screened the best expression hosts for Drosophila-derived retinin protein, and optimized its expression. Chinese hamster ovary (CHO) cells were identified as the efficient expression host of retinin, and purified retinin protein was obtained. At the same time, the preparation method of lanolin nanoemulsion was explored, and the best anti-reflective ability of the nano-coating was determined when the ratio of specific concentration of retinin protein and wax emulsion was 16:4, the pH of the nano-coating formation system was 7.0, and the temperature was 30 ℃. The enhanced antireflective ability and reduced production cost of artificial antireflective nanocoatings by determining the composition of nanocoatings and optimizing the concentration, pH and temperature of system components may facilitate future application of artificial green degradable antireflective coatings.

Recombinant porcine interferon-gamma expressed in CHO cells and its antiviral activity / 生物工程学报

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×107 U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis / 生物工程学报

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

IL-33 gene recombination and expression does not affect the phenotypic characteristics of rabies virus in vitro / 细胞与分子免疫学杂志

Objective To create a recombinant rabies virus overexpressing IL-33 and to clarify the effect of IL-33 overexpression on the phenotypic characteristics of recombinant virus in vitro. Methods The IL-33 gene was obtained and amplified from the brain of a highly virulent strain of rabies infected mouse. It was then inserted between the G and L genes of the parental virus LBNSE genome by reversing genetic manipulation and rescuing a recombinant virus overexpressing IL-33. BSR cells or mouse NA cells were infected with recombinant rabies virus (rLBNSE-IL33) and the parental strain LBNSE. Sequencing and fluorescent antibody virus neutralization assay was employed to detect the stability of recombinant virus at multiplicity of infection=0.01. Viral titres focal forming units (FFU) were detected to plot multi-step growth curves (multiplicity of infection=0.01). Cytotoxicity assay kit was used to detect cellular activity. ELISA was adopted to identify the IL-33 in the supernatant of infected cells of different multiplicity of infection. Results Rescued rLBNSE-IL33 overexpressing IL-33 remained stable for at least 10 consecutive generations and had virus titers of approximately 108 FFU/mL. rLBNSE-IL33 was able to express IL-33 at high levels in a dose-dependent manner, but no high expression of IL-33 was detected in the supernatant of cells infected by LBNSE. Examination of the titers of rLBNSE-IL33 and the parental strain LBNSE in BSR and NA cells over 5 days showed no significant differences and similar kinetic properties in growth. Overexpression of IL-33 had no significant effect on the proliferation and activity of infected cells. Conclusion Overexpression of IL-33 does not significantly affect the phenotypic characteristics of recombinant rabies virus in vitro.

Mitochondrial energy metabolism in baby hamster kidney (BHK-21/C13) cells treated with karnozin extra® / Metabolismo energético mitocondrial en células de riñón de hámster bebé (BHK-21 / C13) tratadas con karnozin extra®

SUMMARY: Carnosine is known as a natural dipeptide, which inhibits the proliferation of tumor cells throughout its action on mitochondrial respiration and cell glycolysis. However, not much is known about its effects on the metabolism of healthy cells. We explored the effects of Karnozin EXTRA® capsule with different concentrations of L-carnosine, on the cell viability and the expressions of intermediate filament vimentin (VIM) and superoxide dismutase (SOD2) in normal fibroblasts BHK-21/C13. Furthermore, we investigated its action on the energy production of these cells. Cell viability was quantified by the MTT assay. The Clark oxygen electrode (Oxygraph, Hansatech Instruments, England) was used to measure the "intact cell respiration rate", state 3 of ADP-stimulated oxidation, maximum oxidation capacity and the activities of complexes I, II and IV. Results showed that Karnozin EXTRA® capsule in concentrations of 2 and 5 mM of L-carnosine did not induce toxic effects and morphological changes in treated cells. Our data revealed a dose-dependent immunofluorescent signal amplification of VIM and SOD2 in the BHK-21/C13 cell line. This supplement substantially increased the recorded mitochondrial respiration rates in the examined cell line. Due to the stimulation of mitochondrial energy production in normal fibroblasts, our results suggested that Karnozin EXTRA® is a potentially protective dietary supplement in the prevention of diseases with altered mitochondrial function.

RESUMEN: La carnosina se conoce como dipéptido natural, que inhibe la proliferación de células tumorales a través de su acción sobre la respiración mitocondrial y la glucólisis celular. Sin embargo, no se sabe mucho de sus efectos sobre el metabolismo de las células sanas. Exploramos los efectos de la cápsula Karnozin EXTRA® con diferentes concentraciones de L-carnosina, sobre la viabilidad celular y las expresiones de vimentina de filamento intermedio (VIM) y superóxido dismutasa (SOD2) en fibroblastos normales BHK-21 / C13. Además, estudiamos su acción sobre la producción de energía de estas células. La viabilidad celular se cuantificó mediante el ensayo MTT. Se utilizó el electrodo de oxígeno Clark (Oxygraph, Hansatech Instruments, Inglaterra) para medir la "tasa de respiración de células intactas", el estado 3 de oxidación estimulada por ADP, la capacidad máxima de oxidación y las actividades de los complejos I, II y IV. Los resultados mostraron que la cápsula de Karnozin EXTRA® en concentraciones de 2 y 5 mM de L- carnosina no indujo efectos tóxicos ni cambios morfológicos en las células tratadas. Nuestros datos revelaron una amplificación de señal inmunofluorescente dependiente de la dosis de VIM y SOD2 en la línea celular BHK-21 / C13. Este suplemento aumentó sustancialmente las tasas de respiración mitocondrial registradas en la línea celular examinada. Debido a la estimulación de la producción de energía mitocondrial en fibroblastos normales, nuestros resultados sugirieron que Karnozin EXTRA® es un suplemento dietético potencialmente protector en la prevención de enfermedades con función mitocondrial alterada.