Reduced Neutralization of SARS-CoV-2 Omicron Variant in Sera from SARS-CoV-1 Survivors after 3-dose of Vaccination

Recent studies found that Omicron variant escapes vaccine-elicited immunity. Interestingly, potent cross-clade pan-sarbecovirus neutralizing antibodies were found in survivors of the infection by SARS-CoV-1 after BNT162b2 mRNA vaccination (N Engl J Med. 2021 Oct 7;385(15):1401-1406). These pan-sarbecovirus neutralizing antibodies were observed to efficiently neutralize the infection driven by the S protein from both SARS-CoV and multiple SARS-CoV-2 variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta). However, whether these cross-reactive antibodies could neutralize the Omicron variant is still unknown. Based on the data collected from a cohort of SARS-CoV-1 survivors received 3-dose of immunization, our studies reported herein showed that a high level of neutralizing antibodies against both SARS-CoV-1 and SARS-CoV-2 were elicited by a 3rd-dose of booster vaccination of protein subunit vaccine ZF2001. However, a dramatically reduced neutralization of SARS-CoV-2 Omicron Variant (B.1.1.529) is observed in sera from these SARS-CoV-1 survivors received 3-dose of Vaccination. Our results indicates that the rapid development of pan-variant adapted vaccines is warranted.

Broad neutralization against SARS-CoV-2 variants induced by a modified B.1.351 protein-based COVID-19 vaccine candidate

Beginning in late 2020, the emergence and spread of multiple variant SARS-CoV-2 strains harboring mutations which may enable immune escape necessitates the rapid evaluation of second generation COVID-19 vaccines, with the goal of inducing optimized immune responses that are broadly protective. Here we demonstrate in a mouse immunogenicity study that two doses of a modified B.1.351 spike (S)-Trimer vaccine (B.1.351 S-Trimer) candidate can induce strong humoral immune responses that can broadly neutralize both the original SARS-CoV-2 strain (Wuhan-Hu-1) and Variants of Concern (VOCs), including the UK variant (B.1.1.7), South African variant (B.1.351) and Brazil variant (P.1). Furthermore, while immunization with two doses (prime-boost) of Prototype S-Trimer vaccine (based on the original SARS-CoV-2 strain) induced lower levels of cross-reactive neutralization against the B.1.351 variant, a third dose (booster) administered with either Prototype S-Trimer or B.1.351 S-Trimer was able to increase neutralizing antibody titers against B.1.351 to levels comparable to neutralizing antibody titers against the original strain elicited by two doses of Prototype S-Trimer.

Broad and differential animal ACE2 receptor usage by SARS-CoV-2

The COVID-19 pandemic has caused an unprecedented global public health and economy crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, comparing to the SARS-CoV-2-like CoVs identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human ACE2 as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2 from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activity, respectively. Among the remaining species, ACE2 from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models. ImportanceSARS-CoV-2 uses human ACE2 as primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologues and found that wild type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models and molecular basis of receptor binding for SARS-CoV-2.

LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

C3A is a sub-clone of human hepatoblastoma HepG2 cell line with the strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ADAP2, GILT and LY6E, three cellular proteins with known activity of interfering virus entry, expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-OC43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFITM3 restriction of human coronavirus entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a distinct mechanism. ImportanceVirus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control by host innate and adaptive immune responses. In the last decade, several interferon inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as host factors to facilitate the entry of several human pathogenic viruses, including human immunodeficiency virus, influenza A virus and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

Improvement of Growth and Periplocin Yield of Periploca sepium Adventitious Root Cultures by Altering Nitrogen Source Supply / 中草药·英文版

Objective To increase the ultimate yield of periplocin in Periploca sepium adventitious root cultures by a two-stage culture based on nitrogen source.Methods Firstly,the effects of nitrogen source(NH-NO-)at different ratios and different total initial nitrogen amounts on the accumulation of biomass and secondary metabolites in adventitious root cultures of P sepium were investigated,and growth and production media for the two-stage culture based on the above results were established.Results The highest biomass and periplocin content were obtained in the culture medium of 15 mmol/L total nitrogen amount with NH-NO(1:2)and 30 mmol/L total nitrogen amount with nitrate as the sole nitrogen source.By adopting a fed-batch cultivation strategy,the dry weight adventitious root,periplocin content and yield were increased by 136%,108%,and 389%,respectively when compared with those of the control,reaching up to 8.13 g/L,157.15 μg/g,and 1277.63 μg/L,respectively.Furthermore,it was found that in the process of two-stage culture,the adventitious roots grew thicker significantly after they were transferred into production medium directly.Conclusion The ultimate yield of periplocin in P.sepium adventitious root cultures could be significantly increased by a two-stage culture based on nitrogen source.

Screening and identification of an ssDNA aptamer targeting the extracellular domain of Mucin16 by CE-SELEX / 医学研究生学报

Objective CA125 has been proved to be closely related to peritoneal metastasis of gastric cancer.This study aimed to screen and identify a novel ssDNA aptamer targeting the extracellular domain of Mucin16. Methods Using capillary elec-trophoresis, we screened the aptamers targeting the synthetic peptide of the extracellular domain of Mucin16 and quantitatively deter-mined the Kd value of each cycle and the affinity of the aptamers for the synthetic peptide by CE-SELEX.Then we evaluated the ability of the obtained aptamers to target cancer cells using confocal laser imaging. Results After five cycles of screening, sequencing, af-finity determination, we obtained an ssDNA aptamer targeting the extracellular domain of Mucin16, with a Kd value of 122.7 nm and visible green fluorescent signals on the cell membrane of the human ovarian cancer cell line expressing Mucin16, but not on that of the normal hepatocytes not expressing Mucin16.This confirmed the binding ability of the aptamer to the extracellular domain of Mucin16. Conclusion A novel aptamer targeting the synthetic peptide of the extracellular domain of Mucin16 was successfully obtained by capil-lary electrophoresis, which could be used as a new agent in the diagnosis and treatment of peritoneal metastasis of gastric cancer.

Improving β-carotene production in Escherichia coli by metabolic engineering of glycerol utilization pathway / 生物工程学报

Glycerol is a byproduct during biodiesel production. It is an important feedstock for fermentation due to its low price and high reduced status. Multiple genes of the glycerol utilization pathway were modulated in a previously engineered high β-carotene producing Escherichia coli strain CAR015 to enhance glycerol utilization capability for improving isoprenoids production. The glpR gene, encoding glycerol 3-phosphate repressor, was firstly deleted. The glpFK, glpD and tpiA genes were then modulated by three artificial regulatory parts, M1-37, M1-46 and M1-93, respectively. β-carotene titer reached 64.82 mg/L after modulating glpD with M1-46, which was 4.86 times higher than that of CAR015, and glycerol consumption rate also increased 100%. Modulating tpiA led to a little increase of β-carotene titer, whereas modulating glpFK led to a little decrease of β-carotene titer. This demonstrated that GlpD was a rate-limiting step in glycerol utilization pathway. Q-PCR of glpF, glpK, glpD and tpiA results showed that decrease the transcription level of glpF, glpK, glpD, or decrease the transcription level of tpiA could increase the cell growth and β-carotene production, probably for the decrease of methylglyoxal toxicity. Modulating glpD and tpiA genes in combination resulted in the best strain Gly003, which produced 72.45 mg/L β-carotene with a yield of 18.65 mg/g dry cell weight. The titer was 5.23 and yield 1.99 times of that of the parent strain CAR015. Our work suggested that appropriate activation of glpD and tpiA genes in glycerol utilization pathway could effectively improve β-carotene production. This strategy can be used for production of other terpenoids in E. coli.

Induction of Periploca sepium in vitro plantlet and studies on dynamic accumulation of periplocin / 中国中药杂志

<p><b>OBJECTIVE</b>To analyze the content of periplocin in different part of the Periploca sepium in vitro plantlet and study its dynamic variation during the process of differentiation.</p><p><b>METHOD</b>The seeds were generated seedling under aseptic condition, and the cut hypocotyl was induced to form the callus and adventitious buds on the MS culture medium with the hormone of IBA 0.1 mg x L(-1) + BA 1 mg x L(-1). The seedling was cut down when the buds grew up to 3 cm and then the root was cultured in the 1/2 MS culture medium with the hormone of IBA 0.5 mg x L(-1) to form intact plantlet. Different parts of it were collected and the content of periplocin was measured during the process of differentiation.</p><p><b>RESULT</b>The contents of periplocin varied widely in different parts during the process of differentiation, with the highest in the roots and then callus, stem and leaf of intact plantlet, stem and leaf of plantlet without root from high to low.</p><p><b>CONCLUSION</b>The periplocin of the secondary metabolite is more likely to be produced and accumulated in root and callus. Periplocin in stem and leaf is probably transported by conducting tissue.</p>