Longitudinal changes in renal function in patients with chronic hepatitis B on antiviral treatment.

BACKGROUND: Patients with chronic hepatitis B (CHB) on nucleos(t)ide analogues (NUCs) often experience renal function decline. Conflicting results regarding the impact of NUC use and renal function have recently been reported. AIM: To examine longitudinal changes in renal function according to the NUC treatment type compared with untreated patients METHODS: From 2014 to 2022, we retrospectively analysed 10,642 patients with CHB. The primary outcome was chronic kidney disease (CKD) progression, which was defined as a minimum one-stage elevation. We applied propensity score (PS) matching for outcome comparisons. RESULTS: In the PS-matched cohort of 1996 pairs, the NUC-treated group (7.6/100 person-years [PYs]) had a significantly higher CKD progression risk than the untreated group (4.4/100 PYs), with a hazard ratio (HR) of 1.70 (p < 0.001). The tenofovir disoproxil fumarate (TDF)-treated group (7.9/100 PYs) showed a 1.76-fold increased CKD progression risk compared with the untreated group (4.5/100 PYs) in the PS-matched cohort (p < 0.001). Both the entecavir- and tenofovir alafenamide (TAF)-treated groups showed CKD progression risks comparable to those of the untreated group in the PS-matched cohorts of 755 and 426 pairs, respectively (p = 0.132 and p = 0.120, respectively). No significant CKD progression risk was found between the entecavir- (6.0/100 PYs) and TAF-treated (5.2/100 PYs) groups in the PS-matched cohort of 510 pairs (p = 0.118). CONCLUSIONS: NUC-treated patients, especially those on TDF, faced a higher CKD progression risk than untreated patients. Entecavir- and TAF-treated patients had comparable CKD progression risks to untreated patients. No difference was observed between entecavir and TAF in the risk of CKD progression.

Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice.

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcKP) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcKP showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-FcM). The animal group injected with EpCAM-FcKP (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-FcM (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.

Expression of Colorectal Cancer Antigenic Protein Fused to IgM Fc in Chinese Cabbage (Brassica rapa).

The epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen and a potential target for tumor vaccine. The EpCAM is a cell-surface glycoprotein highly expressed in colorectal carcinomas. The objective of the present study is to develop an edible vaccine system through Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa). For the transformation, two plant expression vectors containing genes encoding for the EpCAM recombinant protein along with the fragment crystallizable (Fc) region of immunoglobulin M (IgM) and Joining (J)-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K) were constructed. The vectors were successfully transformed and expressed in the Chinese cabbage individually using Agrobacterium. The transgenic Chinese cabbages were screened using genomic polymerase chain reaction (PCR) in T0 transgenic plant lines generated from both transformants. Similarly, the immunoblot analysis revealed the expression of recombinant proteins in the transformants. Further, the T1 transgenic plants were generated by selfing the transgenic plants (T0) carrying EpCAM-IgM Fc and J-chain K proteins, respectively. Subsequently, the T1 plants generated from EpCAM-IgM Fc and J-chain K transformants were crossed to generate F1 plants carrying both transgenes. The presence of both transgenes was validated using PCR in the F1 plants. In addition, the expression of Chinese cabbage-derived EpCAM-IgM Fc × J-chain K was evaluated using immunoblot and ELISA analyses in the F1 plants. The outcomes of the present study can be utilized for the development of a potential anti-cancer vaccine candidate using Chinese cabbage.

Optimization of Ammonium Sulfate Concentration for Purification of Colorectal Cancer Vaccine Candidate Recombinant Protein GA733-FcK Isolated from Plants.

A protein purification procedure is required to obtain high-value recombinant injectable vaccine proteins produced in plants as a bioreactor. However, existing purification procedures for plant-derived recombinant proteins are often not optimized and are inefficient, with low recovery rates. In our previous study, we used 25-30% ammonium sulfate to precipitate total soluble proteins (TSPs) in purification process for recombinant proteins from plant leaf biomass which has not been optimized. Thus, the objective in this study is to optimize the conditions for plant-derived protein purification procedures. Various ammonium sulfate concentrations (15-80%) were compared to determine their effects on TSPs yield. With 50% ammonium sulfate, the yield of precipitated TSP was the highest, and that of the plant-derived colorectal cancer-specific surface glycoprotein GA733 fused to the Fc fragment of human IgG tagged with endoplasmic reticulum retention signal KDEL (GA733(P)-FcK) protein significantly increased 1.8-fold. SDS-PAGE analysis showed that the purity of GA733(P)-FcK protein band appeared to be similar to that of an equal dose of mammalian-derived GA733-Fc (GA733(M)-Fc). The binding activity of purified GA733(P)-FcK to anti-GA733 mAb was as efficient as the native GA733(M)-Fc. Thus, the purification process was effectively optimized for obtaining a high yield of plant-derived antigenic protein with good quality. In conclusion, the purification recovery rate of large quantities of recombinant protein from plant expression systems can be enhanced via optimization of ammonium sulfate concentration during downstream processes, thereby offering a promising solution for production of recombinant GA733-Fc protein in plants.

Effect of the developmental stage and tissue position on the expression and glycosylation of recombinant glycoprotein GA733-FcK in transgenic plants.

The influence of developmental stage and position (top, middle, and base) of leaves and stem tissues on the expression and glycosylation pattern of a recombinant therapeutic protein -GA733-FcK- was observed in transgenic seedlings during a 16-week growth period. RNA expression gradually increased with age in the middle and basal leaves and decreased in top leaves after 14 weeks. The protein expression level at all leaf positions increased until 14 weeks and slightly decreased at 16 weeks; it was lower in yellow leaves than in green leaves. In stem, protein expression gradually decreased from the top to the base. The glycosylation patterns of GA733-FcK were analyzed from 10 to 16 weeks. The plant-specific glycans increased in the top leaves at 14 weeks, but only slightly changed in the middle and basal leaves. The structure of glycans varied with tissue position. The glycosylation level in the top and middle leaves increased until 12 and 14 weeks, respectively, and decreased thereafter, whereas it decreased in basal leaves until 14 weeks and increased at 16 weeks. In stem, all three sections showed high-mannose type glycan structures. The area size of the glycans was significantly higher in the top stem than in both the middle and basal stems, and it was smaller in yellow leaves than in green leaves. The glycan profiles were similar between green and yellow leaves until 16 weeks. Thus, biomass-harvesting time should be optimized to obtain recombinant therapeutic proteins with ideal glycan structure profiles.