A Vaccine Based on the Receptor-Binding Domain of the Spike Protein Expressed in Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses.

In 2020 and 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, caused a global pandemic. Vaccines are expected to reduce the pressure of prevention and control, and have become the most effective strategy to solve the pandemic crisis. SARS-CoV-2 infects the host by binding to the cellular receptor angiotensin converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the surface spike (S) glycoprotein. In this study, a candidate vaccine based on a RBD recombinant subunit was prepared by means of a novel glycoengineered yeast Pichia pastoris expression system with characteristics of glycosylation modification similar to those of mammalian cells. The candidate vaccine effectively stimulated mice to produce high-titer anti-RBD specific antibody. Furthermore, the specific antibody titer and virus-neutralizing antibody (NAb) titer induced by the vaccine were increased significantly by the combination of the double adjuvants Al(OH)3 and CpG. Our results showed that the virus-NAb lasted for more than six months in mice. To summarize, we have obtained a SARS-CoV-2 vaccine based on the RBD of the S glycoprotein expressed in glycoengineered Pichia pastoris, which stimulates neutralizing and protective antibody responses. A technical route for fucose-free complex-type N-glycosylation modified recombinant subunit vaccine preparation has been established.

[Establishment of reflux esophagitis models in rats].

OBJECTIVE: To establish animal models of reflux esophagitis in rats. METHODS: Seventy male Sprague Dawley rats aged 8 weeks were randomly divided into 4 groups: in Group A (n=20) esophagojejunostomy was performed to induce a gastro-jejuno-esophageal reflux; in Group B (n=20) esophagoduodenostomy was performed to induce a gastro-duodeno-esophageal reflux; in Group C (n=20) total gastrectomy plus esophagojejunostomy was performed to induce a jejuno-esophageal reflux; in Group D (n=10) only was performed sham operation (control). RESULT: Among 70 rats, 6 died in Group A, 7 died in Group B, 6 died in Group C, and 72.9 %(51/70) animals were completed in the study. After 12 weeks the incidence of esophageal inflammation was 100.0%; in Groups A, B and C erosion occurred in 11/14 (78.6%), 10/13 (76.9%), 3/14 (21.4%) of animals, respectively; squamous dysplasia was in 10/14 (71.4%), 10/13 (76.9%), 5/14 (35.7%) of rats, respectively; Barrett's esophagus was in 6/14 (42.9%), 5/13 (38.5%), 1/14 (7.1%), respectively. One esophageal adenocarcinoma was found in Group A; no histological changes were observed in Group D. CONCLUSION: The animal models of reflux esophagitis can be induced by esophagojejunostomy, esophagoduodenostomy or total gastrectomy plus esophago-jejunostomy in rats; and the former two surgical modalities are better than the later.

[The effects of nuclear factor-kappaB decoy oligonucleotides on dextran sulphate sodium-induced colitis: experiment with mice].

OBJECTIVE: To investigate the effects of nuclear factor-kappaB (NF-kappaB) decoy oligonucleotide (ODN) on dextran sulphate sodium (DSS)-induced colitis. METHODS: Nine female BABL/C mice underwent infusion of 0.15 ml normal saline into the distant colon and used as controls (Group 1). Twenty-seven female BABL/C mice were made into DSS-induced colitis models and then randomly divided into 3 groups: Group 2 (underwent infusion of 0.15 ml normal saline into the distant colon), Group 3 (infused with NF-kappaB decoy ODN 25 nmol solved in 0.15 ml), and Group 4 (infused with NF-kappaB scrambled decoy ODN 25 nmol solved in 0.15 ml). Disease active index (DAI) was observed every day. Nine days later the mice were killed and their colons were taken out to undergo histological examination. The tumor necrosis factor (TNF)-alpha level of the colon mucosa was measured by enzyme linked immunosorbent assay (ELISA). NF-kappaB expression was determined by immunohistochemical staining. The distribution of NF-kappaB decoy ODN was investigated by confocal laser microscopy. RESULTS: (1) The DAI scores, histological scores and TNF-a level in the colon mucosa of Groups 2 - 4 were all significantly higher than those of Group 1 (all P < 0.05). The DAI scores, histological scores and TNF-a level in the colon mucosa of Group 3 were all significantly lower than those of Groups 2 and 4 (all P < 0.01). (2) In the tissue sections NF-kappaB p65 was positive mainly in the nucleus in the 3 DSS-treated groups without significant differences among these 3 groups, and was mainly positive in the cytoplasm in the control group. (3) Confocal laser microscopy showed that NF-kappaB decoy ODN could be ingested efficiently into the mucosa and submucous layer of colon. (4) There were no significant differences in the liver function, kidney function, and blood glucose among all groups. CONCLUSION: NF-kappaB pathway is associated with the pathogenesis of DSS-induced colitis which is very similar to human UC. Blockade of NF-kappaB pathway by NF-kappaB decoy ODN shows protective effect on the mice with DSS-induced colitis.