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Currently,human health due to corona virus disease 2019(COVID-19)pandemic has been seriously threatened.The coronavirus severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19.However,the efficacy is compromised by the SARS-CoV-2 evolvement and mutation.Here we report the SARS-CoV-2 S protein receptor-binding domain(RBD)inhibitor licorice-saponin A3(A3)could widely inhibit RBD of SARS-CoV-2 variants,including Beta,Delta,and Omicron BA.1,XBB and BQ1.1.Furthermore,A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells,with EC50 of 1.016 pM.The mechanism was related to binding with Y453 of RBD deter-mined by hydrogen-deuterium exchange mass spectrometry(HDX-MS)analysis combined with quan-tum mechanics/molecular mechanics(QM/MM)simulations.Interestingly,phosphoproteomics analysis and multi fluorescent immunohistochemistry(mIHC)respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 mitogen-activated protein kinase(MAPK)path-ways and rebalancing the corresponding immune dysregulation.This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.
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@#Objective To compare the effects of different signal peptides on the secretion and expression of SARS-CoV-2S1,receptor binding domain(RBD) and RBD dimer proteins in Expisf9 insect cells.Methods The gene sequences of three proteins,SARS-CoV-2 S1(M1-E661),RBD(R319-P545) and RBD dimer(R319-K537 tandem),were selected and divided into 25 groups according to the different N-terminal signal peptide sequences(Endo,honeybee melittin(HBM),GP64,GP67,chitinase(Chi) and HIV-ENV) and C-terminal label sequences.25 recombinant baculoviruses were constructed by Bac-to-Bac system,and 25 groups of tertiary strain banks were prepared.B2 and C4 viruses were inoculated to logarithmic prestage cells(2.8 × 10~6 cells/mL) and logarithmic metaphase cells(1.2 × 10~7 cells/mL),respectively.The viruses of each group were cultured to 100 mL(500 mL shaker) for protein expression,and samples were taken for SDSPAGE electrophoresis,Western-blot and ELISA detection.Two groups with higher expression levels of S1,RBD and RBD dimer proteins were selected for repeated verification.Results When B2 and C4 were inoculated to high cell density,the secretion expression level showed no increase,while there were significant difference between 4 and 5 d after inoculation.The expression level of A7(Endo-S1-tag) was significantly lower than that of A9(HIV-ENV-S1-tag),the expression level of A4(Gp67-S1-tag) was the highest,and the secreted expression level of A1(Endo-Endo-Sl-tag) was significantly lower than that of A7(Endo-S1-tag).The secretion and expression of B6(HIV-ENV-RBD-tag) was signifi-cantly higher than that of B4(Gp67-RBD-tag) and other signal peptide groups,and C4(Gp67-RBD-dimer-tag) expression was significantly higher than that of C3(Gp64-RBD-dimer-tag).Two groups with high expression of each protein were selected separately for repeated verification(A4,A9;B4,B6;C3,C4) and the results showed that A4,B6 and C4 had the highest secretion expression levels.Conclusion The signal peptide for the highest secretion expression of S1 and RBD dimer proteins is the same,which is GP67 signal peptide,while the most suitable signal peptide for RBD protein is HIV-ENV,indicating that the N-terminal sequence can affect protein secretion,signal peptide sequence is universal to a certain extent,but is also related to the target protein sequence to be expressed.
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Objective To optimize the immune scheme of SARS-CoV-2 RBD recombinant protein vaccine based on P.pastoris,and investigate the effect of different adjuvants on neutralizating antibody(NAb)titer,in order to provide reference for the continuous optimization research of SARS-CoV-2 vaccine.Methods The RBD protein was selected and the corresponding gene fragment was synthesized,which was constructed into the pPICZαA plasmid,and the plasmid was integrated into the genome of P.pastoris after linear transforma-tion for recombinant expression.The obtained recombinant protein vaccine was combined with different adju-vants to immunize mice to evaluate its immunogenicity.Results Both the target proteins wtRBD and Delta RBD were able to achieve satisfactory overexpression through the P.pastoris system.Compared with the 42 d interval,the IgG antibody titer at the 28 d interval increased by 1.8 times(44 923 vs.80 507).After 3 doses of immunization at an interval of 28 d,the geometric mean titer of NAb for Delta variant was 2.5 times higher than that at an interval of 42 days(2 191 vs.891).After immunization with Delta RBD recombinant protein vaccine combined with aluminum adjuvant,the NAb geometric mean titer for Delta variants reached 32 255(2 167-88 084).When using 5 μg or 30 μg Delta RBD immunization,the NAb titers of the aluminum adju-vant+CpG adjuvant group were about 10 times higher than those of the aluminum adjuvant group alone.Af-ter the third immunization,there was no significant difference in Delta RBD specific IgG titers between the 5 μg antigen group and the 30 μg antigen group(P>0.05).Conclusion Both wtRBD and Delta RBD prepared based on P.pastoris could be used as effective antigens,with three doses of vaccine administered at a 28 day in-terval being the most effective.The combined immunization of Delta RBD recombinant protein with aluminum adjuvant+CpG adjuvant could obtain higher titers of NAb to exert immune effects on SARS-CoV-2 and its va-riants,providing some reference for the continuous optimization research of SARS-CoV-2 vaccines.
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@#Objective To compare the effects of different signal peptides on the secretion and expression of SARS-CoV-2S1,receptor binding domain(RBD) and RBD dimer proteins in Expisf9 insect cells.Methods The gene sequences of three proteins,SARS-CoV-2 S1(M1-E661),RBD(R319-P545) and RBD dimer(R319-K537 tandem),were selected and divided into 25 groups according to the different N-terminal signal peptide sequences(Endo,honeybee melittin(HBM),GP64,GP67,chitinase(Chi) and HIV-ENV) and C-terminal label sequences.25 recombinant baculoviruses were constructed by Bac-to-Bac system,and 25 groups of tertiary strain banks were prepared.B2 and C4 viruses were inoculated to logarithmic prestage cells(2.8 × 10~6 cells/mL) and logarithmic metaphase cells(1.2 × 10~7 cells/mL),respectively.The viruses of each group were cultured to 100 mL(500 mL shaker) for protein expression,and samples were taken for SDSPAGE electrophoresis,Western-blot and ELISA detection.Two groups with higher expression levels of S1,RBD and RBD dimer proteins were selected for repeated verification.Results When B2 and C4 were inoculated to high cell density,the secretion expression level showed no increase,while there were significant difference between 4 and 5 d after inoculation.The expression level of A7(Endo-S1-tag) was significantly lower than that of A9(HIV-ENV-S1-tag),the expression level of A4(Gp67-S1-tag) was the highest,and the secreted expression level of A1(Endo-Endo-Sl-tag) was significantly lower than that of A7(Endo-S1-tag).The secretion and expression of B6(HIV-ENV-RBD-tag) was signifi-cantly higher than that of B4(Gp67-RBD-tag) and other signal peptide groups,and C4(Gp67-RBD-dimer-tag) expression was significantly higher than that of C3(Gp64-RBD-dimer-tag).Two groups with high expression of each protein were selected separately for repeated verification(A4,A9;B4,B6;C3,C4) and the results showed that A4,B6 and C4 had the highest secretion expression levels.Conclusion The signal peptide for the highest secretion expression of S1 and RBD dimer proteins is the same,which is GP67 signal peptide,while the most suitable signal peptide for RBD protein is HIV-ENV,indicating that the N-terminal sequence can affect protein secretion,signal peptide sequence is universal to a certain extent,but is also related to the target protein sequence to be expressed.
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Background & objectives: Vaccination and natural infection can both augment the immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but how omicron infection has affected the vaccine-induced and hybrid immunity is not well studied in Indian population. The present study was aimed to assess the durability and change in responses of humoral immunity with age, prior natural infection, vaccine type and duration with a minimum gap of six months post-two doses with either ChAdOx1 nCov-19 or BBV152 prior- and post-emergence of the omicron variant. Methods: A total of 1300 participants were included in this observational study between November 2021 and May 2022. Participants had completed at least six months after vaccination (2 doses) with either ChAdOx1 nCoV-19 or an inactivated whole virus vaccine BBV152. They were grouped according to their age (? or ?60 yr) and prior exposure of SARS-CoV-2 infection. Five hundred and sixteen of these participants were followed up after emergence of the Omicron variant. The main outcome was durability and augmentation of the humoral immune response as determined by anti-receptor-binding domain (RBD) immunoglobulin G (IgG) concentrations, anti-nucleocapsid antibodies and anti-omicron RBD antibodies. Live virus neutralization assay was conducted for neutralizing antibodies against four variants – ancestral, delta and omicron and omicron sublineage BA.5. Results: Before the omicron surge, serum anti-RBD IgG antibodies were detected in 87 per cent participants after a median gap of eight months from the second vaccine dose, with a median titre of 114 [interquartile range (IQR) 32, 302] BAU/ml. The levels increased to 594 (252, 1230) BAU/ml post- omicron surge (P<0.001) with 97 per cent participants having detectable antibodies, although only 40 had symptomatic infection during the omicron surge irrespective of vaccine type and previous history of infection. Those with prior natural infection and vaccination had higher anti-RBD IgG titre at baseline, which increased further [352 (IQR 131, 869) to 816 (IQR 383, 2001) BAU/ml] (P<0.001). The antibody levels remained elevated after a mean time gap of 10 months, although there was a decline of 41 per cent. The geometric mean titre was 452.54, 172.80, 83.1 and 76.99 against the ancestral, delta, omicron and omicron BA.5 variants in the live virus neutralization assay. Interpretation & conclusions: Anti-RBD IgG antibodies were detected in 85 per cent of participants after a median gap of eight months following the second vaccine dose. Omicron infection probably resulted in a substantial proportion of asymptomatic infection in the first four months in our study population and boosted the vaccine-induced humoral immune response, which declined but still remained durable over 10 months
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@#ObjectiveTo construct and identify a recombinant adenovirus expressing S protein receptor binding domain(RBD)and N protein of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)Delta variant.MethodsThe RBD and N gene fragments of SARS-CoV-2 were cloned into pcDNA3.0BA vector respectively to construct recombinant plasmid pcDNA3.0BA-RBD-N. The RBD-CMV-N fragment was amplified by PCR and inserted into shuttle vector pShuttle-CMV. The shuttle plasmid pShuttle-RBD-N was then homologously recombined with pAdeasy-1 to obtain recombinant plasmid pAdeasy-1-RBD-N,which was transfected into HEK293 cells for recombinant adenovirus Ad-RBD-N packaging. The transcription of RBD and N genes of recombinant adenovirus in HEK293 cells was detected by RT-PCR,while the expre-ssion of RBD and N proteins by Western blot and immunofluorescence assay. 12 female BALB/c mice were immunized with Ad-RBD-N by intramuscular injection at a dose of 5 × 109copies per mouse. Blood samples were collected 14 d after immunization,and the serum antibody titers were measured by ELISA.ResultsThe RBD and N genes of recombinant adenovirus were transcribed normally in HEK293 cells,and the RBD and N proteins were expressed normally in MA104 cells. Mice immunized with the recombinant adenovirus produced specific IgG antibodies against RBD and N proteins.ConclusionThe recombinant adenovirus expressing S protein RBD and N protein of SARS-CoV-2 Delta variant was succe-ssfully constructed,which laid a foundation of the follow-up research on Delta variant vaccines.
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Yersinia pestis phage is a virus that is parasitic within Yersinia pestis and can specifically lyses Yersinia pestis. The adsorption sites of phage infesting host bacteria are called receptor binding protein (RBP), including extracellular membrane protein, lipopolysaccharide, teichoteic acid, pili, flagella, capsular polysaccharide, etc., of which extracellular membrane protein and lipopolysaccharide are the receptors of Yersinia pestis phage. RBP plays a decisive role in the process of Yersinia pestis phage infecting Yersinia pestis. Therefore, the classification, isolation and application of Yersinia pestis phage are summarized; the research progress in identification and structure of Yersinia pestis phage receptor is analyzed, which is helpful in understanding the cleavage mechanism of Yersinia pestis phage and the interaction mode with Yersinia pestis from the molecular level, and provide more powerful support for in-depth study on Yersinia pestis phage receptor.
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@#Objective To construct a single-chain fragment variable(scFv)phage display library against receptor-binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(S)to screen specific scFv and identify the function.Methods m RNA was extracted from spleen cells of mice immunized with RBD protein and reversely transcribed into c DNA,with which as template,genes of the hight chain fragment of variable(VH)and light chain fragment of variable(VL)of scFv were amplified and then assembled into scFv gene fragment through splicing overlap extension PCR(SOE-PCR).The scFv gene fragment was inserted to phage vector to construct scFv phage display library.After four rounds of biopanning,the scFv gene with strong binding ability to RBD was screened and expressed recombinantly,purified and identified for biological activity.Results The constructed scFv phage library showed a titer of 6.0×10(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(334~353)),RBD9(S(334~353)),RBD9(S(439~458))and RBD13(S(439~458))and RBD13(S(499~518)).Homologous model of scFv constructed by online server SWISS-MODEL showed a good quality and was used for molecular docking.The interface at which scFv11 interacted with RBD only partially coincided with the interaction interface of human angiotensin converting enzyme 2(ACE2)and RBD,and the interaction interfaces of scFv12 and scFv25 with RBD were quite different from that of ACE2.Conclusion In this study,scFv specifically bound to SARS-Co V-2 RBD was screened and prepared through constructing scFv phage library against SARS-CoV-2 RBD,which provided experimental basis for further development of anti-SARS-CoV-2 drugs and detection reagents.
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【Objective】 To investigate the effect of immunoglobulin G (IgG) dimer concentration of intravenous immunoglobulin (IVIG) on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Methods】 Firstly, protein purification and high performance liquid chromatography (HPLC) were used to prepare different concentrations of IgG dimers. After that, IgG dimer was added to IVIG to prepare IVIG containing different concentrations of IgG dimer. Finally, based on the method established in our laboratory, we analyzed the effect of IgG dimer concentration in IVIG on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Results】 When the concentration of IgG dimer in IVIG was 1.11%-10.30%, its binding ability to Fc receptors on the surface of THP-1 cell was 97.67%-135.33%, and this binding ability was positively correlated with the concentration of IgG dimer. When the IgG dimer concentration exceeded 13.22%, the binding ability had no correlation with the IgG dimer concentration. 【Conclusion】 A certain concentration of IgG dimer can promote the binding ability of the IgG Fc fragment in IVIG to receptors on the surface of THP-1 cells, which needs further verification from animal experiments and clinical data.
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@#Abstract: Objective To construct SARS-CoV-2 receptor binding domain molecular probe for monoclonal memory B cell sorting and obtain RBD specific neutralizing antibodies from peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescents by single-cell sorting. Methods The SARS-CoV-2 RBD sequence was downloaded from GenBank, and the Avi tag and 6-histidine tags were added at the C-terminal. After codon optimization, it was chemically synthesized, cloned into the pDRVI1.0 vector, expressed after transfection of 293F cells, and biotinylated consequently. RBD-specific B cells were sorted out with this probe1 from the PBMCs of convalescents recovered from COVID-19. After B cells were lysed, the variable regions of heavy chain and light chain were amplified, cloned into the antibody expression vector, and transfected into 293F cells to express the antibody. Then the antibody was purified from the supernatant using protein A column and SARS-CoV-2 pseudovirus was used to test their neutralizing activity. Results RBD-Avi probe was produced and successfully biotinylated sequentially with an efficiency of 30%-50%. Western blot analysis revealed that the biotinylated probe was recognized by the antibodies purified from COVID-19 convalescent plasma. Using this probe, 7 and 16 RBD-specific memory B cells were successfully isolated from the PBMCs of two convalescent individuals, accounting for 0.24% and 0.17% of the total cell population, respectively. After amplifying the variable regions of antibody heavy and light chains from the lysed B cells, 7 and 12 pairs of antibody heavy-light chains were obtained. A total of 16 antibodies were expressed in the convalescent individuals, and most of the purified antibodies showed neutralizing activity against the pseudovirus, with IC50 values of 6 antibodies below 1 μg/mL. The IC50 values of XJ-A9 and SCF-F1 against the wild-type pseudovirus were 0.07 μg/mL and 0.35 μg/mL, respectively. Conclusion The SARS-CoV-2 RBD molecular probe constructed in this study has good antigenicity, and the isolated antibodies present neutralizing activity against wild-type SARS-CoV-2 pseudovirus.
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Porcine epidemic diarrhea (PED) is a highly contagious disease that causes high mortality in suckling piglets. Although several licensed inactivated and live attenuated vaccines were widely used, the infection rate remains high due to unsatisfactory protective efficacy. In this study, mRNA vaccine candidates against PED were prepared, and their immunogenicity was evaluated in mice and pregnant sows. The mRNA PED vaccine based on heterodimer of viral receptor binding region (RBD) showed good immunogenicity. It elicited robust humoral and cellular immune responses in mice, and the neutralizing antibody titer reached 1:300 after a single vaccination. Furthermore, it induced neutralizing antibody level similar to that of the inactivated vaccine in pregnant sows. This study developed a new design of PED vaccine based on the mRNA-RBD strategy and demonstrated the potential for clinical application.
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Grossesse , Animaux , Femelle , Souris , Suidae , Anticorps antiviraux , Maladies des porcs/épidémiologie , Vaccins antiviraux/génétique , Anticorps neutralisants , Vaccins atténués , Diarrhée/médecine vétérinaireRésumé
BACKGROUND The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can infect common mice inducing significant pathological lung lesions and inflammatory responses. This substantially mimics coronavirus disease 19 (COVID-19) infection and pathogenesis in humans. OBJECTIVES To characterise the effects of recombinant SARS-CoV-2 S1 receptor-binding domain (RBD) peptide in murine macrophage and microglial cells' immune activation compared with classical PAMPs in vitro. METHODS Murine RAW 264.7 macrophages and BV2 microglial cells were exposed to increasing concentrations of the RBD peptide (0.01, 0.05, and 0.1 µg/mL), Lipopolysaccharide (LPS) and Poly(I:C) and evaluated after two and 24 h for significant markers of macrophage activation. We determined the effects of RBD peptide on cell viability, cleaved caspase 3 expressions, and nuclear morphometry analysis. FINDINGS In RAW cells, RBD peptide was cytotoxic, but not for BV2 cells. RAW cells presented increased arginase activity and IL-10 production; however, BV2 cells expressed iNOS and IL-6 after RBD peptide exposure. In addition, RAW cells increased cleaved-caspase-3, apoptosis, and mitotic catastrophe after RBD peptide stimulation but not BV2 cells. CONCLUSION RBD peptide exposure has different effects depending on the cell line, exposure time, and concentration. This study brings new evidence about the immunogenic profile of RBD in macrophage and microglial cells, advancing the understanding of SARS-Cov2 immuno- and neuropathology.
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ObjectiveTo investigate the specific anti-SARS-CoV-2 antibody in adults and above after initial vaccination with inactivated COVID-19 vaccine, and determine the influencing factors. MethodsIn this study, residents aged 18 and above who had completed two doses of inactivated COVID-19 vaccine in Deqing County, Huzhou City, Zhejiang Province were included. Information such as gender, age, type of vaccine and vaccination time were collected, and serum specimens were sampled. Anti-SARS-CoV-2 receptor binding domain (RBD) antibody was quantitatively examined by enzyma-linked immunosorbent assay (ELISA) and influencing factors were determined. ResultsThe median concentration of anti-SARS-CoV-2 IgG antibody in the residents vaccinated with an inactivated booster vaccine was higher than that in those vaccinated with only two doses of COVID-19 vaccine or single dose (P<0.05). The median concentration of IgG antibody in males was 9.73 (4.01‒23.70) RU‧mL-1, lower than 17.76 (7.07‒49.23) RU‧mL-1 in females (P<0.05). The median concentration in the residents vaccinated with BBIBP-CorV (Sinopharm) was 6.53 (0.97‒13.69) RU‧mL-1, which was lower than that in those vaccinated with CoronaVac (Sinovac) that was 17.29 (8.54‒43.73) RU‧mL-1 (P<0.05). The median concentration in those with BBIBP-CorV was also lower than 12 (5.45‒40.06) RU‧mL-1 in those with heterologous booster vaccine (P<0.05). The median concentration was 9.73 (3.83‒23.63) RU‧mL-1 in the residents with an interval of more than 6 months from the second dose, which was lower than 14.66 (6.36‒35.98) RU‧mL-1 in those with an interval of 3‒6 months (P<0.05). Moreover, immune effect was better in females (χ²=16.464, P<0.05), 18‒45 years(χ²=7.158, P<0.05), and those vaccinated with CornaVac (χ²=49.637, P<0.05), while decreased in those with an interval of more than 6 months from the second dose (χ²=8.447, P<0.05). ConclusionGender, age, and type of vaccine may affect the effect of immunization. The COVID-19 vaccination shows an acceptable immunogenicity in adults; however, it declines in 6 months after vaccination. It warrants strengthening the booster vaccination to maintain the immune response.
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Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.
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Objective:To investigate the efficacy of a SARS-CoV-2 recombinant protein vaccine as a booster dose.Methods:A new immunogen, namely RBD-sc-trimer, was designed by tandem repeating of single receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein to mimic the trimeric form of RBD presented by the virus. The RBD-sc-trimer protein was expressed as a His-tagged fusion protein using a baculovirus expression system and purified by nickel affinity column. The purified protein was identified by Western blot. Its in vitro binding activity to human angiotensin converting enzyme 2 (hACE2) was analyzed by ELISA. The immunogenicity of RBD-sc-trimer as well as RBD proteins of other forms including RBD dimer (RBD-Fc), RBD monomer (RBD) and S protein trimer (S trimer) as a booster dose was evaluated in BALB/c mice. Results:In terms of both binding and neutralizing antibodies against SARS-CoV-2, RBD-sc-trimer showed an immunogenicity that was superior to that of RBD-Fc and RBD and close to the level of S trimer. The antibody response induced by RBD-sc-trimer was characterized as Th1-biased. Moreover, it displayed a stronger cross-neutralization activity against SARS-CoV-2 Beta, Delta and Omicron variants. The titer of neutralizing antibody against Omicron induced by RBD-sc-trimer only decreased by 9.1 folds relative to the prototype strain, while the antibody response induced by RBD-Fc and S trimer decreased by 68.4 and 70.8 folds, respectively.Conclusions:The recombinant protein, RBD-sc-trimer, which was capable of eliciting stronger humoral response in mice as a booster dose and showed the superiority in raising cross-reactive antibodies against SARS-CoV-2 variants over non-trimeric RBD forms, should be considered as an optimal immunogen for the development of more effective SARS-CoV-2 vaccines.
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The COVID-19 pandemic is still spreading around the world,posing a severe challenge to public health security and economic development in China and beyond. The pivotal process of SARS-COV-2 invasion is that the receptor binding domain (RBD) of Coronavirus spike protein binds with the angioten-sin converting enzyme 2 (ACE2) receptor on host cells. Therefore, RBD is closely related to our major anti-epidemic strategies such as rapid pathogen detection and development of vaccines and antiviral drugs. In this study, we aim to compare the anti-RBD IgG by immunizing mice with the recombinant RBDs of novel Coronavirus spike protein, and evaluate the antibody titers and duration elicited by RBD produced from different host cells and used in different doses, which can be used for vaccine evaluation, drug development, and pathogen detection. SDS-PAGE and mass spectrometry analysis showed that the molecular weight of bRBD (produced by insect cells) and hRBD (produced by mammalian cells) were slightly different, which are mainly caused by different glycosylation modification. Flow cytometry revealed that the binding rates of bRBD and hRBD to HEK293-ACE2-overexpressing cells were 88. 5% and 92. 7%, respectively, indicating that both antigens have favorable bioactivity. Balb/ c mice are immunized intramuscularly with 10 μg and 20 μg per mouse of RBD proteins, which were mixed with Quick Antibody adjuvant previously, and blood samples collected from the tail at 2, 4, 6, 8, 12 and 24 weeks after the primary immunization. Enzyme-linked immunosorbent assay (ELISA) showed that both RBD immunization with 10 μg or 20 μg could induce a rapid immune response to produce IgG antibodies. The antibody titers reached the peak at 4-6 weeks post first immunization, and persisted over a long time up to 6 months. No significant difference was observed in the induced antibody titers by bRBD and hRBD as well as their different doses. Virus Neutralization Assays showed that specific antibodies induced by RBD-immunized mice could inhibit the infection of ACE2-positive cells by SARS-CoV-2 pseudovirus. These results suggest that both RBD proteins through commendable adjuvant inoculation can rapidly induce an effective humoral immune response and generate specific neutralizing antibodies. It is expected to be helpful for rapid preparation of antibodies against the future various RBD mutants during the pandemic.
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Bacteriophages bind to the bacteria receptor through the receptor binding proteins (RBPs), a process that requires the involvement of complex atomic structures and conformational changes. In response to bacteriophage infection, bacteria have developed a variety of resistance mechanisms, while bacteriophages have also evolved multiple antagonistic mechanisms to escape host resistance. The exploration of the "adsorption-anti adsorption-escape process" between bacteriophages and bacteria helps us better understand the co-evolution process of bacteriophages and bacteria, which is important for the development of phage therapeutic technologies and phage-based biotechnologies. This review summarizes the bacteriophage adsorption related proteins, how bacteriophages escape host resistance based on the RBP alternations, and the recent progress of RBP-related biotechnologies.
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Bactéries , Récepteurs de bactériophages , Bactériophages/génétique , Protéines de transport , Liaison aux protéinesRésumé
The behavioral responses exerted by spinal administration of the opioid-neurotensin hybrid peptide, PK23, were studied in adult male rats. The antinociceptive effect upon exposure to a thermal stimulus, as well as tolerance development, was assessed in an acute pain model. The PK23 chimera at a dose of 10 nmol/rat produced a potent pain-relieving effect, especially after its intrathecal administration. Compared with intrathecal morphine, this novel compound was found to possess a favourable side effect profile characterized by a reduced scratch reflex, delayed development of analgesic tolerance or an absence of motor impairments when given in the same manner, though some animals died following barrel rotation as a result of its i.c.v. administration (in particular at doses higher than 10 nmol/rat). Nonetheless, these results suggest the potential use of hybrid compounds encompassing both opioid and neurotensin structural fragments in pain management. This highlights the enormous potential of synthetic neurotensin analogues as promising future analgesics.
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Objective • To study the effects of different phosphorylation levels of nuclear receptor-binding factor 2 (NRBF2) on the activity of class III phosphatidylinositol 3-kinase complex (PI3KC3-C1), and find a type of PI3KC3-C1 with the highest kinase activity as a target for the screening of autophagy-specific inhibitors. Methods • First, in vitro protein purification system was established to obtain quaternary NRBF2-free PI3KC3-C1 and quinary PI3KC3-C1 containing different phosphorylation levels of NRBF2. The integrity of the complex was determined by gel filtration chromatography. The different kinase activity of purified PI3KC3-C1 was detected in vitro, and thus the effect of phosphorylation of NRBF2 on PI3KC3-C1 activity was observed. Results • The purified PI3KC3-C1 had good purity and yield. The five-membered PI3KC3-C1 containing NRBF2 showed higher kinase activity than the quaternary protein complex, and PI3KC3-C1 containing the continuous dephosphorylated NBRF2 had the highest kinase activity. Conclusion • NRBF2 does exist as one of the components of PI3KC3-C1. The presence of NRBF2 promotes the kinase activity of PI3KC3-C1, and PI3KC3-C1 containing continuous dephosphorylation of NRBF2 may serve as a new regulatory target for the screening of autophagy-specific inhibitor.
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Objective To establish an indirect enzyme-linked immunosorbent assay (ELISA)and to compare the efficiency of receptor binding domain (RBD) proteins in different forms for Middle East re-spiratory syndrome coronavirus (MERS-CoV) antibody detection. Methods The monomeric and trimeric forms of MERS-CoV RBD were expressed in Bac-insect cells, 293T cells and ExpiCHO-STM expression sys-tem and then purified. The purified RBD proteins were identified with native gel electrophoresis and Western blot. Then, an equal amount of each RBD protein was used as coating antigen to establish an ELISA for de-tecting MERS-CoV IgG titer. For comparison, the newly developed ELISA and the commercial MERS-CoV IgG antibody detection kit (Euroimmune with S1 as the coating antigen) were used to measure the MERS-CoV antibody reference panel supplied by World Health Organization (WHO). Results The purified mon-omeric and trimeric MERS-CoV RBD were successfully prepared using 293T cells and ExpiCHO-STM system. RBD antigens of different forms and from different systems could recognize MERS-CoV specific antibody without having any cross reaction with the sera from healthy adults. The in-house RBD-based ELISA had good detection consistency with the Euroimmune commercial kit. The positive samples showed higher and more concentrated values based on the RBD trimer than the monomer. Conclusions Novel indirect ELISA methods based on the monomeric and trimeric forms of RBD protein were established. The trimetric form-based ELISA achieved higher detection efficiency than the one using the monomer antigen, suggesting that it could be uses as a competent alternative to the commercial kit.