Objective To investigate whether Omicron BA.1 breakthrough infection after receiving the SARS-CoV-2 vaccine could create a strong immunity barrier.Methods Blood samples were collected at two different time points from...Objective To investigate whether Omicron BA.1 breakthrough infection after receiving the SARS-CoV-2 vaccine could create a strong immunity barrier.Methods Blood samples were collected at two different time points from 124 Omicron BA.1 breakthrough infected patients and 124 controls matched for age,gender,and vaccination profile.Live virus-neutralizing antibodies against five SARS-CoV-2 variants,including WT,Gamma,Beta,Delta,and Omicron BA.1,and T-lymphocyte lymphocyte counts in both groups were measured and statistically analyzed.Results The neutralizing antibody titers against five different variants of SARS-CoV-2 were significantly increased in the vaccinated population infected with the Omicron BA.1 variant at 3 months after infection,but mainly increased the antibody level against the WT strain,and the antibody against the Omicron strain was the lowest.The neutralizing antibody level decreased rapidly 6 months after infection.The T-lymphocyte cell counts of patients with mild and moderate disease recovered at 3 months and completely returned to the normal state at 6 months.Conclusion Omicron BA.1 breakthrough infection mainly evoked humoral immune memory in the original strain after vaccination and hardly produced neutralizing antibodies specific to Omicron BA.1.Neutralizing antibodies against the different strains declined rapidly and showed features similar to those of influenza.Thus,T-lymphocytes may play an important role in recovery.展开更多
Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in...Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.展开更多
Mutations in the receptor binding domain(RBD)of SARS-CoV-2 spike protein have given birth to the new variants of concern viz.Delta and Omicron strain.As we are using vaccines which are either mRNA of spike protein of ...Mutations in the receptor binding domain(RBD)of SARS-CoV-2 spike protein have given birth to the new variants of concern viz.Delta and Omicron strain.As we are using vaccines which are either mRNA of spike protein of Wuhan strain or its whole virion vaccine,these vaccines will trigger antibodies to be able to bind with the epitopes of spike protein of Wuhan strain.There appears a need to study whether vaccine generated circulating neutralizing antibodies in vaccinated people are able to neutralize the receptor binding domain of spike protein of currently circulating Delta and Omicron strains as well.We need to address this contention in two phases:first to undertake docking studies of vaccine generated antibodies with Wuhan,Delta and Omicron strains and second to conduct the wet lab studies and observe antigen-antibody bindings employing Immunofluorescence and Cryo-Electron Microscopic studies.The present paper reports results of docking studies using bioinformatic softwares to provide the baseline information for wet lab studies.展开更多
AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injecti...AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injection of macrophage-rich peritoneal exudate cells(PECs) and the local expression of TGF-β isoforms was assessed by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA) at various time points.In addition,we investigated the effect of TFG-β-neutralizing antibodies(TGF-β NAb) on subretinal fibrosis development.· RESULTS:TGF-β1 and TGF-β2 mRNA level was significantly elevated at day 2 after subretinal fibrosis induction and increased further to 5 and 6.5-fold respectively at day 5,reaching the peak.TGF-β3 mRNA was not detected in the present study.The result of ELSIA showed that active TGF-β1 and TGF-β2 levels were upregulated to 10-fold approximately,while total TGF-β1 and TGF-β2 levels were even upregulated more than 10-fold and more than 20-fold respectively in subretinal fibrosis mice in comparison with na?觙ve mice at day 5.TGF-β NAb resulted in a reduced subretinal fibrosis areas by 65% compared to animals from control group at day 7.· CONCLUSION:Our results indicate that TGF-β signaling may contribute to the pathogenesis of subretinal fibrogenesis and TGF-β inhibition may provide an effective,novel treatment of advanced and late-stage neovascular age-related macular degeneration.展开更多
The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome ...The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)outbreak.Using these tools,we can assess the presence and duration of antibody-mediated protection in naturally infected individuals,screen convalescent plasma preparations for donation,test the efficacy of immunotherapy,and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects.This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.展开更多
Stromal cell-derived factor-1 and its receptor CXCR4 are essential regulators of the neurogenesis that occurs in the adult hippocampal dentate gyrus.However,the effects of CXCR7,a new atypical receptor of stromal cell...Stromal cell-derived factor-1 and its receptor CXCR4 are essential regulators of the neurogenesis that occurs in the adult hippocampal dentate gyrus.However,the effects of CXCR7,a new atypical receptor of stromal cell-derived factor-1,on hippocampal neurogenesis after a stroke remain largely unknown.Our study is the first to investigate the effect of a CXCR7-neutralizing antibody on neurogenesis in the dentate gyrus and the associated recovery of cognitive function of rats in the chronic stage of cerebral ischemia.The rats were randomly divided into sham,sham+anti-CXCR7,ischemia and ischemia+anti-CXCR7 groups.Endothelin-1 was injected in the ipsilateral motor cortex and striatum to induce focal cerebral ischemia.Sham group rats were injected with saline instead of endothelin-1 via intracranial injection.Both sham and ischemic rats were treated with intraventricular infusions of CXCR7-neutralizing antibodies for 6 days 1 week after surgery.Immunofluorescence staining with doublecortin,a marker for neuronal precursors,was performed to assess the neurogenesis in the dentate gyrus.We found that anti-CXCR7 antibody infusion enhanced the proliferation and dendritic development of doublecortin-labeled cells in the dentate gyrus in both ischemic and sham-operated rats.Spatial learning and memory functions were assessed by Morris water maze tests 30-32 days after ischemia.CXCR7-neutralizing antibody treatment significantly reduced the escape latency of the spatial navigation trial and increased the time spent in the target quadrant of spatial probe trial in animals that received ischemic insult,but not in sham operated rats.These results suggest that CXCR7-neutralizing antibody enhances the neurogenesis in the dentate gyrus and improves the cognitive function after cerebral ischemia in rats.All animal experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of China Medical University(CMU16089 R)on December 8,2016.展开更多
The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) ...The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.展开更多
The ongoing coronavirus disease 2019(COVID-19)pandemic is a global public health crisis,causing social and economic disasters in many countries.In China,two-consecutive negative results of nucleic acid tests for SARS-...The ongoing coronavirus disease 2019(COVID-19)pandemic is a global public health crisis,causing social and economic disasters in many countries.In China,two-consecutive negative results of nucleic acid tests for SARS-CoV-2 from the respiratory samples are required to end the quarantine of COVID-19 patients.However,clinicians face a dilemma in case of patients with long-term viral shedding.This report described an unusual COVID-19 case who had persistent viral RNA positivity for more than 4 months after initial illness in the presence of low neutralizing antibodies,but without prolonged clinical symptoms.Multiple anti-viral drug treatments had no impact and there was no evidence of re-infection.When the patient was self-quarantined at home,no infection occurred to the three family members living with her for 15 to 19 days.Sputum viral culture in BSL-3 laboratory on the 102nd day after symptom onset was negative.From the 129th day on,8 continuous nucleic acid tests of sputum samples showed negative results.The patient was discharged on 137th days since symptom onset.In conclusion,viral RNA shedding in the sputum of the COVID-19 patient may last over 4 months.As no evidence shows the existence of infectious virus,two-consecutive negative nucleic acid tests may not be the prerequisite for ending quarantine of COVID-19 patients with prolonged viral shedding.展开更多
Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for han...Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.展开更多
Objective The coronavirus disease 2019(COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody(NAb) responses ...Objective The coronavirus disease 2019(COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody(NAb) responses to severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods In total, 605 serum samples from 125 COVID-19 patients(from January 1 to March 14, 2020)varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2.Results NAbs were detectable approximately 10 days post-onset(dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective.Conclusion We demonstrated that NAb levels vary among all categories of COVID-19 patients. Longterm studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.展开更多
Coronavirus disease 2019 is threatening thousands of millions of people around the world.In the absence of specific and highly effective medicines,the treatment of infected persons is still very challenging.As therape...Coronavirus disease 2019 is threatening thousands of millions of people around the world.In the absence of specific and highly effective medicines,the treatment of infected persons is still very challenging.As therapeutics,neutralizing antibodies(NAbs)have great potential.Many NAbs have been reported,and most target various regions on the receptor-binding domain of the spike(S)protein,or the N-terminal domain.Several NAbs and NAb cocktails have been authorized for emergency use,and more arc in clinical trials or are under development.In this review,considering the angle of binding epitopes on the S protein,we summarize the functions and the underlying mechanisms of a set of well-recognized NAbs and provide guidance for vaccine design and the combinatorial use of these antibodies.In addition,we review the NAbs and NAb cocktails that have been approved for emergency use and discuss the effectiveness of these NAbs for combating severe acute respiratory syndrome coronavirus 2 mutants.展开更多
Smallpox eradication was successful via prophylactic administration of live attenuated vaccinia virus. As a result of the discontinuation of the smallpox immunization program, many individuals are now susceptible to s...Smallpox eradication was successful via prophylactic administration of live attenuated vaccinia virus. As a result of the discontinuation of the smallpox immunization program, many individuals are now susceptible to smallpox virus infection should it be used as a biological weapon. Presently, only individuals at high risk for exposure are required to receive smallpox vaccine, such as laboratory personnel that handle variola/vaccinia virus. This study endeavored to investigate a one-year period of vaccinia virus-specific T cell responses using polychromatic flow cytometry and neutralizing (Nt) antibody responses using plaque reduction neutralization test (PRNT) in individuals receiving primary immunization (n = 5) with ACAM2000<sup>TM</sup> smallpox vaccine. Functional and phenotypic profiles of vaccinia virus-specific T cell responses were characterized. Each single functional measurement {CD107a/b expression, production of interferon g (IFN-g), macrophage inflammatory protein 1b (MIP-1b), interleukin 2 (IL-2), and tumor necrosis factor a (TNF-a)} demonstrated that vaccinia virus-specific CD8<sup>+</sup> T cells were functional at least one time point after vaccination (p ≤ 0.05). However, vaccinia virus-specific CD4<sup>+</sup> T cells were functional only for MIP-1b production (p ≤ 0.05). Vaccinia virus-specific CD8<sup>+</sup> T cells induced in these individuals showed increased polyfunctionality in at least 2 phenotypes relative to pre-vaccination (p ≤ 0.05). Although only three of five individuals (60%) showed positive Nt antibody (titer ≥ 20) at first month after vaccination, all five individuals (100%) demonstrated Nt antibody at 2 months, post-immunization. Interestingly, all vaccinees could retain the Nt antibody for 6 months after primary vaccination. In conclusion, ACAM2000<sup>TM</sup> smallpox vaccine induced both polyfunctional T cell-and Nt antibody-responses in primary immunized individuals.展开更多
A one-dimensional(1 D) self-organized array composed of dislocation and anti-dislocation is analytically investigated in the frame of Peierls theory. From the exact solution of the Peierls equation, it is found that t...A one-dimensional(1 D) self-organized array composed of dislocation and anti-dislocation is analytically investigated in the frame of Peierls theory. From the exact solution of the Peierls equation, it is found that there exists strong neutralizing effect that makes the Burgers vector of each individual dislocation in the equilibrium array smaller than that of an isolated dislocation. This neutralizing effect is not negligible even though dislocations are well separated. For example, when the distance between the dislocation and the anti-dislocation is as large as ten times of the dislocation width, the actual Burgers vector is only about 80% of an isolated dislocation. The neutralizing effect originates physically from the power-law asymptotic behavior that enables two dislocations interfere even though they are well separated.展开更多
Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased...Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010.S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV.In this study,the construction,expression and purification of Pseudomonas aeruginosa exotoxin A(PE)without domain Ⅲ(PE△Ⅲ)as a vector was performed for the delivery of PEDV S-A or S-B.PE(△Ⅲ)PEDV S-A and PE(△Ⅲ)PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice.The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses,but also stimulate PEDV-specific mucosal immune responses in mice.PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.展开更多
Objective To investigate neutralizing antibody levels in symptomatic and asymptomatic patients with coronavirus disease 2019(COVID-19)at 6 and 10 months after disease onset.Methods Blood samples were collected at thre...Objective To investigate neutralizing antibody levels in symptomatic and asymptomatic patients with coronavirus disease 2019(COVID-19)at 6 and 10 months after disease onset.Methods Blood samples were collected at three different time points from 27 asymptomatic individuals and 69 symptomatic patients infected with severe acute respiratory syndrome coronavirus 2(SARS-Co V-2).Virus-neutralizing antibody titers against SARS-CoV-2 in both groups were measured and statistically analyzed.Results The symptomatic and asymptomatic groups had higher neutralizing antibodies at 3 months and 1–2 months post polymerase chain reaction confirmation,respectively.However,neutralizing antibodies in both groups dropped significantly to lower levels at 6 months post-PCR confirmation.Conclusion Continued monitoring of symptomatic and asymptomatic individuals with COVID-19 is key to controlling the infection.展开更多
Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Met...Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.展开更多
Despite extensive research efforts, a preventive human immunodeficiency virus (HIV) vaccine remains one of the major challenges in the field of AIDS research. Experimental strategies which have been proven successful ...Despite extensive research efforts, a preventive human immunodeficiency virus (HIV) vaccine remains one of the major challenges in the field of AIDS research. Experimental strategies which have been proven successful for other viral vaccines are not enough to tackle HIV-1 and new approaches to design effective preventive AIDS vaccines are of utmost importance. Due to enormous diversity among global circulating HIV strains, an effective HIV vaccine must elicit broadly protective antibodies based responses;therefore discovering new broadly neutralizing antibodies (bNAbs) against HIV has become major focus in HIV vaccine research. However further understanding of the viral targets of such antibodies and mechanisms of action of bNAbs is required for advancement of HIV vaccine research. This technical note discusses our current knowledge on the bNAbs and immunoprophylaxis using viral vectors with their relevance in designing of new candidates to HIV-1 vaccines.展开更多
In this present study, we predicted the neutralizing epitope of a modeled H5N1 hemagglutinin 1046T when interacted with a modeled monoclonal antibody variable fragment 8H5Fv using molecular dynamics simulation. Follow...In this present study, we predicted the neutralizing epitope of a modeled H5N1 hemagglutinin 1046T when interacted with a modeled monoclonal antibody variable fragment 8H5Fv using molecular dynamics simulation. Following the production run of the molecular dynamics simulation, we observed the average change of solvent accessible surface of the antigen alongside the formation of hydrogen bonds between the two structures during the simulation. Based on the acquired data, we predicted the neutralizing epitope of the 1046T antigen to be consisted of residues Asp 84, Glu85, Phe86, Ile87, Asn88, Val89, Pro90, Ile132, Ser136, Val147, Pro152, Tyr153, Leu154, Arg161, and Tyr268. By calculating the RMSD of the Cα backbone chain of the complex during the simulation we found the structure to be generally stable suggesting a well maintained steric hindrance, while RMSD calculation of the predicted neutralizing epitope backbone suggests the stability of the neutralizing epitope itself.展开更多
The frequency and the consequences of binding and neutralizing antibodies (BAbs and NAbs) against Interferon beta (IFNbeta) in Turkish multiple sclerosis (MS) patients have not been determined yet, which could differ ...The frequency and the consequences of binding and neutralizing antibodies (BAbs and NAbs) against Interferon beta (IFNbeta) in Turkish multiple sclerosis (MS) patients have not been determined yet, which could differ in such a country which is between Europa and Asia. The aim of the study is to assess the frequency of these antibodies, and to evaluate the impact of NAbs, from the clinical and radiologic aspects in Turkish patients with MS. One hundred and two MS patients were included. BAbs were screened using capture enzyme-linked immunosorbent assay (cELISA), and NAbs were detected via Myxovirus protein A (MxA) messenger RNA (mRNA) induction assay (real-time polymerase chain reaction-PCR) at the beginning and one year later. Relapse rate and expanded disability status scale (EDSS) were used to assess the clinical impact. Gadolinium enhanced lesions and T2 lesion volume were used as magnetic resonance imaging (MRI) parameters. Persistent NAb positivity defines to be positive both at first and then one year later. NAbs were detected in 12.2% (6/49) of IFNbeta-1b treated patients, and in 7.5% (3/40) of IFNbeta-1a SC treated patients, but none of the IFNbeta-1a IM treated patients had detectable NAbs. It was found that the mean relapse rate difference was significantly higher in persistent NAb negative patients (p = 0.024). Persistent NAb positivity had no effect on T2 lesion volume and contrast enhancing lesions. 60% of the persistent NAb positive patients had at least one relapse during one-year of follow-up. On the other hand, 32% of persistent NAb negative patients were detected to have at least one relapse. Data from this study suggest that patients may become unresponsive to IFNbeta therapy even when the frequency of NAbs does not prove to be as high as those in the literature. Nevertheless, one should keep in mind that disease activity is not always equal to NAb positivity.展开更多
Objective:To isolate and characterize Staphylococcus aureus(S.aureus)β-hemolysinneutralizing dAbs from phage display library of Indian desert camel.Methods:Phage display library of 5×10 dAb clones of LPS-immuniz...Objective:To isolate and characterize Staphylococcus aureus(S.aureus)β-hemolysinneutralizing dAbs from phage display library of Indian desert camel.Methods:Phage display library of 5×10 dAb clones of LPS-immunized Indian desert camel constructed in our laboratory was used for selection of S.aureus exotoxin-specific clones by panning technique.Enrichment of Ag-specific clones in successive rounds of panning was assessed by phage-ELISA and phage titration.Different dAb clones binding to S.aureus exotoxin Ags were expressed with C-terminal 6×His tag in E.coli and purified by Ni-chelate chromatography.The expression was verified by SDS-PAGE and western blotting.The purified clones were tested for inhibition of ’hot-cold’ hemolytic activity in vitro.Resistance to thermal inactivation of the dAb clones was studied by observing the effect of heat treatment from 50℃to 99℃for 30 min on the ’hot-cold’ hemolytic activity in vitro.Results:Several dAb clones binding to S.aureus exotoxins were isolated and enriched by three rounds of panning.The soluble dAb clones were approximately～16 kDa in size and reacted with 6×His tag specific murine monoclonal antibody in western blot.One of the Ni-chelate affinity purified dAb.6×His clones,inhibited S.aureusβ-hemolysin activity in vitro and resisted thermal inactivation upto 991.Conclusions:An S.aureusβ-hemolysinneutralizing dAb clone of possible therapeutic potential has been isolated.展开更多
基金funded by the Emergency prevention and cure Program of COVID-19[22ZXGBSY00010]Tianjin Medical Key Discipline Project[TJYXZDXK-50A]sponsored by Tianjin Municipal Science and Technology Bureau and Tianjin Municipal Health Commission,respectively.
文摘Objective To investigate whether Omicron BA.1 breakthrough infection after receiving the SARS-CoV-2 vaccine could create a strong immunity barrier.Methods Blood samples were collected at two different time points from 124 Omicron BA.1 breakthrough infected patients and 124 controls matched for age,gender,and vaccination profile.Live virus-neutralizing antibodies against five SARS-CoV-2 variants,including WT,Gamma,Beta,Delta,and Omicron BA.1,and T-lymphocyte lymphocyte counts in both groups were measured and statistically analyzed.Results The neutralizing antibody titers against five different variants of SARS-CoV-2 were significantly increased in the vaccinated population infected with the Omicron BA.1 variant at 3 months after infection,but mainly increased the antibody level against the WT strain,and the antibody against the Omicron strain was the lowest.The neutralizing antibody level decreased rapidly 6 months after infection.The T-lymphocyte cell counts of patients with mild and moderate disease recovered at 3 months and completely returned to the normal state at 6 months.Conclusion Omicron BA.1 breakthrough infection mainly evoked humoral immune memory in the original strain after vaccination and hardly produced neutralizing antibodies specific to Omicron BA.1.Neutralizing antibodies against the different strains declined rapidly and showed features similar to those of influenza.Thus,T-lymphocytes may play an important role in recovery.
基金supported by the Jiangsu Provincial Key Research and Development Program (Grant No.BE2020616)the National Key R&D Program of China (Grant No.2018YFC1200603)+1 种基金the National Science and Technology Major Project (Grant No.2019SWAQ05-5-4)Jiangsu Key Lab of Cancer Biomarkers,Prevention and Treatment,Collaborative Innovation Center for Cancer Personalized Medicine,Nanjing Medical University.
文摘Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.
基金funded by Indian Council of Medical Research,Grant Number 2021-6369.
文摘Mutations in the receptor binding domain(RBD)of SARS-CoV-2 spike protein have given birth to the new variants of concern viz.Delta and Omicron strain.As we are using vaccines which are either mRNA of spike protein of Wuhan strain or its whole virion vaccine,these vaccines will trigger antibodies to be able to bind with the epitopes of spike protein of Wuhan strain.There appears a need to study whether vaccine generated circulating neutralizing antibodies in vaccinated people are able to neutralize the receptor binding domain of spike protein of currently circulating Delta and Omicron strains as well.We need to address this contention in two phases:first to undertake docking studies of vaccine generated antibodies with Wuhan,Delta and Omicron strains and second to conduct the wet lab studies and observe antigen-antibody bindings employing Immunofluorescence and Cryo-Electron Microscopic studies.The present paper reports results of docking studies using bioinformatic softwares to provide the baseline information for wet lab studies.
文摘AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injection of macrophage-rich peritoneal exudate cells(PECs) and the local expression of TGF-β isoforms was assessed by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA) at various time points.In addition,we investigated the effect of TFG-β-neutralizing antibodies(TGF-β NAb) on subretinal fibrosis development.· RESULTS:TGF-β1 and TGF-β2 mRNA level was significantly elevated at day 2 after subretinal fibrosis induction and increased further to 5 and 6.5-fold respectively at day 5,reaching the peak.TGF-β3 mRNA was not detected in the present study.The result of ELSIA showed that active TGF-β1 and TGF-β2 levels were upregulated to 10-fold approximately,while total TGF-β1 and TGF-β2 levels were even upregulated more than 10-fold and more than 20-fold respectively in subretinal fibrosis mice in comparison with na?觙ve mice at day 5.TGF-β NAb resulted in a reduced subretinal fibrosis areas by 65% compared to animals from control group at day 7.· CONCLUSION:Our results indicate that TGF-β signaling may contribute to the pathogenesis of subretinal fibrogenesis and TGF-β inhibition may provide an effective,novel treatment of advanced and late-stage neovascular age-related macular degeneration.
基金supported by grants from the Applied Basic Research Key Project of Wuhan Municipal Bureau of Science and Technology(2020020601012218)the Fundamental Research Funds for the Central Universities(HUST COVID-19 Rapid Response Call No.2020kfyXGYJ040).
文摘The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)outbreak.Using these tools,we can assess the presence and duration of antibody-mediated protection in naturally infected individuals,screen convalescent plasma preparations for donation,test the efficacy of immunotherapy,and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects.This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.
基金supported by the National Natural Science Foundation of China,No.81401002(to SSZ)
文摘Stromal cell-derived factor-1 and its receptor CXCR4 are essential regulators of the neurogenesis that occurs in the adult hippocampal dentate gyrus.However,the effects of CXCR7,a new atypical receptor of stromal cell-derived factor-1,on hippocampal neurogenesis after a stroke remain largely unknown.Our study is the first to investigate the effect of a CXCR7-neutralizing antibody on neurogenesis in the dentate gyrus and the associated recovery of cognitive function of rats in the chronic stage of cerebral ischemia.The rats were randomly divided into sham,sham+anti-CXCR7,ischemia and ischemia+anti-CXCR7 groups.Endothelin-1 was injected in the ipsilateral motor cortex and striatum to induce focal cerebral ischemia.Sham group rats were injected with saline instead of endothelin-1 via intracranial injection.Both sham and ischemic rats were treated with intraventricular infusions of CXCR7-neutralizing antibodies for 6 days 1 week after surgery.Immunofluorescence staining with doublecortin,a marker for neuronal precursors,was performed to assess the neurogenesis in the dentate gyrus.We found that anti-CXCR7 antibody infusion enhanced the proliferation and dendritic development of doublecortin-labeled cells in the dentate gyrus in both ischemic and sham-operated rats.Spatial learning and memory functions were assessed by Morris water maze tests 30-32 days after ischemia.CXCR7-neutralizing antibody treatment significantly reduced the escape latency of the spatial navigation trial and increased the time spent in the target quadrant of spatial probe trial in animals that received ischemic insult,but not in sham operated rats.These results suggest that CXCR7-neutralizing antibody enhances the neurogenesis in the dentate gyrus and improves the cognitive function after cerebral ischemia in rats.All animal experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of China Medical University(CMU16089 R)on December 8,2016.
基金supported by grants from National Science and Technology Major Project(2012ZX10004701)
文摘The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.
文摘The ongoing coronavirus disease 2019(COVID-19)pandemic is a global public health crisis,causing social and economic disasters in many countries.In China,two-consecutive negative results of nucleic acid tests for SARS-CoV-2 from the respiratory samples are required to end the quarantine of COVID-19 patients.However,clinicians face a dilemma in case of patients with long-term viral shedding.This report described an unusual COVID-19 case who had persistent viral RNA positivity for more than 4 months after initial illness in the presence of low neutralizing antibodies,but without prolonged clinical symptoms.Multiple anti-viral drug treatments had no impact and there was no evidence of re-infection.When the patient was self-quarantined at home,no infection occurred to the three family members living with her for 15 to 19 days.Sputum viral culture in BSL-3 laboratory on the 102nd day after symptom onset was negative.From the 129th day on,8 continuous nucleic acid tests of sputum samples showed negative results.The patient was discharged on 137th days since symptom onset.In conclusion,viral RNA shedding in the sputum of the COVID-19 patient may last over 4 months.As no evidence shows the existence of infectious virus,two-consecutive negative nucleic acid tests may not be the prerequisite for ending quarantine of COVID-19 patients with prolonged viral shedding.
文摘Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.
基金the Guangdong Provincial Novel Coronavirus Scientific and Technological Project[2020111107001 and202008040004]the National Key Research and Development Program of China[2020YFC0846900]+1 种基金the Guangzhou Basic Research Program on People’s Livelihood Science and Technology[202002020005]the National Natural Science Foundation of China[82003487 and 82072265]。
文摘Objective The coronavirus disease 2019(COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody(NAb) responses to severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods In total, 605 serum samples from 125 COVID-19 patients(from January 1 to March 14, 2020)varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2.Results NAbs were detectable approximately 10 days post-onset(dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective.Conclusion We demonstrated that NAb levels vary among all categories of COVID-19 patients. Longterm studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.
基金supported by the National Natural Science Foundation of China(Nos.31970130,31600672,31670831,and 31370813).
文摘Coronavirus disease 2019 is threatening thousands of millions of people around the world.In the absence of specific and highly effective medicines,the treatment of infected persons is still very challenging.As therapeutics,neutralizing antibodies(NAbs)have great potential.Many NAbs have been reported,and most target various regions on the receptor-binding domain of the spike(S)protein,or the N-terminal domain.Several NAbs and NAb cocktails have been authorized for emergency use,and more arc in clinical trials or are under development.In this review,considering the angle of binding epitopes on the S protein,we summarize the functions and the underlying mechanisms of a set of well-recognized NAbs and provide guidance for vaccine design and the combinatorial use of these antibodies.In addition,we review the NAbs and NAb cocktails that have been approved for emergency use and discuss the effectiveness of these NAbs for combating severe acute respiratory syndrome coronavirus 2 mutants.
文摘Smallpox eradication was successful via prophylactic administration of live attenuated vaccinia virus. As a result of the discontinuation of the smallpox immunization program, many individuals are now susceptible to smallpox virus infection should it be used as a biological weapon. Presently, only individuals at high risk for exposure are required to receive smallpox vaccine, such as laboratory personnel that handle variola/vaccinia virus. This study endeavored to investigate a one-year period of vaccinia virus-specific T cell responses using polychromatic flow cytometry and neutralizing (Nt) antibody responses using plaque reduction neutralization test (PRNT) in individuals receiving primary immunization (n = 5) with ACAM2000<sup>TM</sup> smallpox vaccine. Functional and phenotypic profiles of vaccinia virus-specific T cell responses were characterized. Each single functional measurement {CD107a/b expression, production of interferon g (IFN-g), macrophage inflammatory protein 1b (MIP-1b), interleukin 2 (IL-2), and tumor necrosis factor a (TNF-a)} demonstrated that vaccinia virus-specific CD8<sup>+</sup> T cells were functional at least one time point after vaccination (p ≤ 0.05). However, vaccinia virus-specific CD4<sup>+</sup> T cells were functional only for MIP-1b production (p ≤ 0.05). Vaccinia virus-specific CD8<sup>+</sup> T cells induced in these individuals showed increased polyfunctionality in at least 2 phenotypes relative to pre-vaccination (p ≤ 0.05). Although only three of five individuals (60%) showed positive Nt antibody (titer ≥ 20) at first month after vaccination, all five individuals (100%) demonstrated Nt antibody at 2 months, post-immunization. Interestingly, all vaccinees could retain the Nt antibody for 6 months after primary vaccination. In conclusion, ACAM2000<sup>TM</sup> smallpox vaccine induced both polyfunctional T cell-and Nt antibody-responses in primary immunized individuals.
基金Project supported by the National Natural Science Foundation of China(Grant No.11874093)
文摘A one-dimensional(1 D) self-organized array composed of dislocation and anti-dislocation is analytically investigated in the frame of Peierls theory. From the exact solution of the Peierls equation, it is found that there exists strong neutralizing effect that makes the Burgers vector of each individual dislocation in the equilibrium array smaller than that of an isolated dislocation. This neutralizing effect is not negligible even though dislocations are well separated. For example, when the distance between the dislocation and the anti-dislocation is as large as ten times of the dislocation width, the actual Burgers vector is only about 80% of an isolated dislocation. The neutralizing effect originates physically from the power-law asymptotic behavior that enables two dislocations interfere even though they are well separated.
基金This research was funded by the National Natural Science Foundation of China(31900746 and 31802170)Postdoctoral Science Foundation(2019M660182).
文摘Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010.S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV.In this study,the construction,expression and purification of Pseudomonas aeruginosa exotoxin A(PE)without domain Ⅲ(PE△Ⅲ)as a vector was performed for the delivery of PEDV S-A or S-B.PE(△Ⅲ)PEDV S-A and PE(△Ⅲ)PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice.The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses,but also stimulate PEDV-specific mucosal immune responses in mice.PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.
基金supported by the National Natural Science Foundation of China[82041027]National Key R&D Program of China[2021ZD0114100,2021ZD0114103]+2 种基金The Capital Health Development and Research of Special[2022-1G-3014]Beijing Science and Technology Planning Project of Beijing Science and Technology Commission[Z211100002521015,Z211100002521019]Cooperation project[2022-jk-cd-009]。
文摘Objective To investigate neutralizing antibody levels in symptomatic and asymptomatic patients with coronavirus disease 2019(COVID-19)at 6 and 10 months after disease onset.Methods Blood samples were collected at three different time points from 27 asymptomatic individuals and 69 symptomatic patients infected with severe acute respiratory syndrome coronavirus 2(SARS-Co V-2).Virus-neutralizing antibody titers against SARS-CoV-2 in both groups were measured and statistically analyzed.Results The symptomatic and asymptomatic groups had higher neutralizing antibodies at 3 months and 1–2 months post polymerase chain reaction confirmation,respectively.However,neutralizing antibodies in both groups dropped significantly to lower levels at 6 months post-PCR confirmation.Conclusion Continued monitoring of symptomatic and asymptomatic individuals with COVID-19 is key to controlling the infection.
基金supported by 2021 Beijing Key Specialty Program for Major Epidemic Prevention and Control。
文摘Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.
文摘Despite extensive research efforts, a preventive human immunodeficiency virus (HIV) vaccine remains one of the major challenges in the field of AIDS research. Experimental strategies which have been proven successful for other viral vaccines are not enough to tackle HIV-1 and new approaches to design effective preventive AIDS vaccines are of utmost importance. Due to enormous diversity among global circulating HIV strains, an effective HIV vaccine must elicit broadly protective antibodies based responses;therefore discovering new broadly neutralizing antibodies (bNAbs) against HIV has become major focus in HIV vaccine research. However further understanding of the viral targets of such antibodies and mechanisms of action of bNAbs is required for advancement of HIV vaccine research. This technical note discusses our current knowledge on the bNAbs and immunoprophylaxis using viral vectors with their relevance in designing of new candidates to HIV-1 vaccines.
文摘In this present study, we predicted the neutralizing epitope of a modeled H5N1 hemagglutinin 1046T when interacted with a modeled monoclonal antibody variable fragment 8H5Fv using molecular dynamics simulation. Following the production run of the molecular dynamics simulation, we observed the average change of solvent accessible surface of the antigen alongside the formation of hydrogen bonds between the two structures during the simulation. Based on the acquired data, we predicted the neutralizing epitope of the 1046T antigen to be consisted of residues Asp 84, Glu85, Phe86, Ile87, Asn88, Val89, Pro90, Ile132, Ser136, Val147, Pro152, Tyr153, Leu154, Arg161, and Tyr268. By calculating the RMSD of the Cα backbone chain of the complex during the simulation we found the structure to be generally stable suggesting a well maintained steric hindrance, while RMSD calculation of the predicted neutralizing epitope backbone suggests the stability of the neutralizing epitope itself.
文摘The frequency and the consequences of binding and neutralizing antibodies (BAbs and NAbs) against Interferon beta (IFNbeta) in Turkish multiple sclerosis (MS) patients have not been determined yet, which could differ in such a country which is between Europa and Asia. The aim of the study is to assess the frequency of these antibodies, and to evaluate the impact of NAbs, from the clinical and radiologic aspects in Turkish patients with MS. One hundred and two MS patients were included. BAbs were screened using capture enzyme-linked immunosorbent assay (cELISA), and NAbs were detected via Myxovirus protein A (MxA) messenger RNA (mRNA) induction assay (real-time polymerase chain reaction-PCR) at the beginning and one year later. Relapse rate and expanded disability status scale (EDSS) were used to assess the clinical impact. Gadolinium enhanced lesions and T2 lesion volume were used as magnetic resonance imaging (MRI) parameters. Persistent NAb positivity defines to be positive both at first and then one year later. NAbs were detected in 12.2% (6/49) of IFNbeta-1b treated patients, and in 7.5% (3/40) of IFNbeta-1a SC treated patients, but none of the IFNbeta-1a IM treated patients had detectable NAbs. It was found that the mean relapse rate difference was significantly higher in persistent NAb negative patients (p = 0.024). Persistent NAb positivity had no effect on T2 lesion volume and contrast enhancing lesions. 60% of the persistent NAb positive patients had at least one relapse during one-year of follow-up. On the other hand, 32% of persistent NAb negative patients were detected to have at least one relapse. Data from this study suggest that patients may become unresponsive to IFNbeta therapy even when the frequency of NAbs does not prove to be as high as those in the literature. Nevertheless, one should keep in mind that disease activity is not always equal to NAb positivity.
基金Financial support by Department of Biotechnology,Government of India,New Delhi for construction of phage display library and its applications, and Indian Council of Agricultural Research,New Delhi for infrastructure assistance
文摘Objective:To isolate and characterize Staphylococcus aureus(S.aureus)β-hemolysinneutralizing dAbs from phage display library of Indian desert camel.Methods:Phage display library of 5×10 dAb clones of LPS-immunized Indian desert camel constructed in our laboratory was used for selection of S.aureus exotoxin-specific clones by panning technique.Enrichment of Ag-specific clones in successive rounds of panning was assessed by phage-ELISA and phage titration.Different dAb clones binding to S.aureus exotoxin Ags were expressed with C-terminal 6×His tag in E.coli and purified by Ni-chelate chromatography.The expression was verified by SDS-PAGE and western blotting.The purified clones were tested for inhibition of ’hot-cold’ hemolytic activity in vitro.Resistance to thermal inactivation of the dAb clones was studied by observing the effect of heat treatment from 50℃to 99℃for 30 min on the ’hot-cold’ hemolytic activity in vitro.Results:Several dAb clones binding to S.aureus exotoxins were isolated and enriched by three rounds of panning.The soluble dAb clones were approximately～16 kDa in size and reacted with 6×His tag specific murine monoclonal antibody in western blot.One of the Ni-chelate affinity purified dAb.6×His clones,inhibited S.aureusβ-hemolysin activity in vitro and resisted thermal inactivation upto 991.Conclusions:An S.aureusβ-hemolysinneutralizing dAb clone of possible therapeutic potential has been isolated.
