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@#Objective To express the Gn protein of severe fever with thrombocytopenia syndrome virus(SFTSV) through adeno-associated virus 9(AAV9) expression system and evaluate its immunogenicity.Methods SFTSV Gn gene was inserted into viral vector pAAV-CMV-FH and the recombinant plasmid was transfected into HEK293T cells to obtain recombinant virus AAV9-Gn.The expression of Gn protein was determined by immunofluorescence and Western blot.Eighteen fernale BALB/c mice were randomly divided into three groups:Mock group(serum-free DMEM),AAV9-GFP group(1 × 10~(11) vg) and AAV9-Gn group(1 × 10~(11) vg),all of which were injected intramuscularly into the right hind limb at a dose of 100 μL per mouse.The body mass,diet,behavior and mental state of mice in each group were monitored continuously for 21 d,and the change rate of body mass was calculated;At 2,4,8 and 16 weeks after immunization,the levels of SFTSV neutralizing antibody in serum of mice in each group were detected by fluorescent reduction neutralization test(FRNT),and the levels of specific IgGl and IgG2a in serum of mice in AAV9-Gn group were detected by ELISA.Results After incubation with specific antibody,Vero cells transfected with AAV9-Gn showed specific green fluorescence under fluorescence microscope,and had specific binding to mouse anti-SFTSV Gn monoclonal antibody,and the specific binding bands were found at a relative molecular mass of about 61 000.The body mass of the three groups showed an increasing trend,there was no significant difference between the three groups(F=0.158—2.621,P> 0.05),and the diet,behavior and mental state were normal.At 2,4,8 and 16 weeks after immunization,the titer of SFTSV neutralizing antibody in serum of mice in AAV9-Gn group was significantly higher than that of Mock group and AAV9-GFP group(H=13.332—14.538,each P <0.001),and the titer peak appeared at 8 weeks;The level of specific IgG1 in serum of mice was significantly higher than that of IgG2a(F=4.373—12.975,each P <0.05) at different time points.Conclusion SFTSV Gn protein can be expressed correctly through AAV9 expression system,and has low toxicity to mice with good immunogenicity,which is expected to be a candidate component of SFTSV vaccine.
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Objective:To establish an antibody expression system to reduce the antibody-dependent enhancement (ADE) effect of target antibody.Methods:Site-directed mutagenesis was used to mutate the 234 and 235 sites of the Fc region of the mammalian cell antibody expression vector-L234A and L235A to establish the antibody expression vector pFRT-IgG1κ-FcM. An antibody Wt-WNV with significant ADE effect obtained in previous work was selected and expressed by the pFRT-IgG1κ-FcM system to obtain mutant antibody FcM-WNV. The binding ability of FcM-WNV to target antigen West Nile virus envelope protein-DⅢ (WNV E-DⅢ) was detected by ELISA, and the its binding ability to human high-affinity IgG Fc receptor hFcγRⅠ (hCD64 ) was analyzed by flow cytometry. The neutralizing activity of FcM-WNV in vitro was detected by pseudovirus infection of host cells (BHK21 and K562). Results:The expression levels of FcM-WNV and Wt-WNV were comparable, and FcM-WNV could recognize and bind to WNVE-DIII in a concentration-dependent manner. Compared with Wt-WNV, the binding ability of FcM-WNV to hCD64 was significantly weakened, showing a significant decrease in fluorescence intensity. Consistent with the previous experimental results, Wt-WNV at a concentration of 5 μg/ml significantly enhanced the infection of K562 by WNV pseudovirus, while FcM-WNV at a concentration of 5 μg/ml could effectively block pseudovirus infection in both K562 and BHK21 cells.Conclusions:The established antibody expression system can effectively reduce the ADE effect of the target antibody.
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Objective:To prepare rabbit polyclonal antibodies against respiratory syncytial virus (RSV) N protein and use them as the detection antibodies to establish a N-ELISA-based method for rapid detection of neutralizing antibodies.Methods:A plasmid of pET30a-N for the expression of RSV N protein was constructed. After purification, the protein was immunized into New Zealand rabbits to prepare polyclonal antibodies, which were used as the detection antibodies. Positive serum samples were diluted and used to neutralize RSV (100 TCID 50/well). Hep-2 cells were inoculated and cultured, and then the cells were fixed with 80% acetone. ELISA was performed to detect RSV N protein in infected cells. When the absorbance value of a well was below the cut-off value, it was regarded as the positive well in the neutralization test. The highest dilution of a positive well serum was the neutralizing antibody titer. After optimizting the antibody dilution, detection time, cell density and the duration of neutralization, the method for neutralizing antibody detection was established based on N-ELISA. The established method was verified by analyzing the influences of different cell generations and edge effects, and calculating the accuracy, repeatability and precision. The correlation between the established method and microneutralization method was analyzed by detecting human RSV IgG-positive serum. Results:The plasmid pET30a-N was successfully constructed, and the expressed N protein showed high purity and good specificity. After the third immunization, the antibody titer in rabbit serum was 1∶51 200, and the antibodies could specifically bind to RSV. The prepared rabbit anti-RSV N polyclonal antibodies had a titer of 1∶51 200, and showed good specificity. The neutralizing antibodies could be detected on day 4 with the established method, and the duration of neutralization was shortened to 30 min. Cell generations and the position of wells in the 96-well plate (edge well and non-edge well) had no significant effect on the method, and the repeatability, precision and accuracy of the method were good. In the detection of 64 RSV IgG-positive human serum samples by the established method and microneutralization method, the correlation coefficient was 0.929 6, indicating a good positive correlation between the two methods.Conclusions:A N-ELISA-based method for rapid neutralizing antibody detection is successfully established, which can be used to evaluate the serum antibody level after RSV vaccination.
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@#Objective To evaluate the immunogenicity of prototype strain,Beta strain,Gamma strain and Delta strain of SARS-CoV-2 inactivated vaccines in rats.Methods Five female Wistar rats were immunized with SARS-CoV-2 inactivated vaccines of prototype,Beta,Gamma and Delta strains through thigh muscle twice at an interval of 14 d,with an immunization dose of 3 μg virus protein/(0.5 mL per rat).Serum samples were collected and isolated by vein 14,28 and 42 d after the first immunization.The serum IgG antibody levels were detected by indirect ELISA,the titers of serum neutralizing antibody were measured by microneutralization,and the antigenic ratios of the serum neutralizing antibody titers were calculated to evaluate the antigenicity difference between different strains.Results at 14 d after the first immunization,IgG antibodies against four strains of virus were detected in all immunized serum samples.The levels of IgG antibodies increased by more than 10 times at 28 d compared with those at 14 d,and decreased slightly at 42 d.At 14 d after the first immunization,all the neutralizing antibodies against the four strains were positive in the serum of rats immunized with prototype strain or Delta strain vaccine;In the serum samples of rats immunized with Beta and Gamma strains,all the neutralizing antibodies against Beta and Gamma strains were positive,while some neutralizing antibodies against prototype or Delta strains were positive.At 28 d after the first immunization,the neutralizing antibodies in the immune serum of the four strains were positive,and the titers of neutralizing antibodies were significantly higher than those at 14 d;The neutralizing antibody titers were slightly lower at 42 d after the first immunization than 28 d.There was small difference in the antigenicity between Beta and Gamma,prototype and Gamma,but significant difference in the antigenicity between prototype and Beta strains.Conclusion The prototype strain,Beta strain,Gamma strain and Delta strain of SARS-CoV-2 inactivated vaccines can stimulate rats to produce neutralizing antibodies with high titer,while the immunogenicity has difference.
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@#Objective To express the Gn protein of severe fever with thrombocytopenia syndrome virus(SFTSV) through adeno-associated virus 9(AAV9) expression system and evaluate its immunogenicity.Methods SFTSV Gn gene was inserted into viral vector pAAV-CMV-FH and the recombinant plasmid was transfected into HEK293T cells to obtain recombinant virus AAV9-Gn.The expression of Gn protein was determined by immunofluorescence and Western blot.Eighteen fernale BALB/c mice were randomly divided into three groups:Mock group(serum-free DMEM),AAV9-GFP group(1 × 10~(11) vg) and AAV9-Gn group(1 × 10~(11) vg),all of which were injected intramuscularly into the right hind limb at a dose of 100 μL per mouse.The body mass,diet,behavior and mental state of mice in each group were monitored continuously for 21 d,and the change rate of body mass was calculated;At 2,4,8 and 16 weeks after immunization,the levels of SFTSV neutralizing antibody in serum of mice in each group were detected by fluorescent reduction neutralization test(FRNT),and the levels of specific IgGl and IgG2a in serum of mice in AAV9-Gn group were detected by ELISA.Results After incubation with specific antibody,Vero cells transfected with AAV9-Gn showed specific green fluorescence under fluorescence microscope,and had specific binding to mouse anti-SFTSV Gn monoclonal antibody,and the specific binding bands were found at a relative molecular mass of about 61 000.The body mass of the three groups showed an increasing trend,there was no significant difference between the three groups(F=0.158—2.621,P> 0.05),and the diet,behavior and mental state were normal.At 2,4,8 and 16 weeks after immunization,the titer of SFTSV neutralizing antibody in serum of mice in AAV9-Gn group was significantly higher than that of Mock group and AAV9-GFP group(H=13.332—14.538,each P <0.001),and the titer peak appeared at 8 weeks;The level of specific IgG1 in serum of mice was significantly higher than that of IgG2a(F=4.373—12.975,each P <0.05) at different time points.Conclusion SFTSV Gn protein can be expressed correctly through AAV9 expression system,and has low toxicity to mice with good immunogenicity,which is expected to be a candidate component of SFTSV vaccine.
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@#Objective To develop and verify a method for detecting the activity of neutralizing antibodies in ELISA antibody positive serum of rats immunized with recombinant human interleukin-1 receptor antagonist(rhIL-1Ra). Methods The SD rats were subcutaneously immunized with 3,20 and 100 mg/kg rhIL-1Ra injection respectively,10 rats in each group,half male and half female,twice a day at an interval of at least 4 h between each dose for 13 consecutive weeks. The blood samples were collected from the jugular vein of rats during the administration period and the recovery period. The serum samples were isolated and detected for the antibody titers by ELISA,and the samples positive for rhIL-1Ra antibody were purified by Protein A chromatographic column. Based on,D10G4·1 cells biological activity assay,a method for the detection of neutralizing antibody activity was developed and verified for the specificity,sensitivity and precision. The neutralizing antibody activity of rhIL-1Ra antibody positive serum determined by ELISA was detected by using the developed method.Results With the increase of doses,the serum antibody titers of rats in various dose groups gradually increased,and there were still antibodies in the recovery period,and the titer was still high. Rabbit anti-rhIL-1Ra monoclonal antibody showed obvious neutralizing effect on rIL-1Ra,while rabbit anti-rIFN-2b monoclonal antibody had no dose-effect relationship with rIL-1Ra. The sensitivity of the method was 171. 93 μg/mL;The CVs of precision verification were not more than 20%. The positive antibody sera detected by ELISA all had neutralizing effect on rhIL-1Ra injection,which was consistent with the results detected by ELISA. Conclusion The method developed in this study has good specificity and high sensitivity in the detection of serum neutralizing antibody activity in rats immunized with rhIL-1Ra,which can be used to detect the serum neutralizing antibody activity of animals with rhIL-1Ra repeated administration.
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The World Health Organization has declared that the outbreak of coronavirus disease 2019(COVID-19) is a global pandemic. As mutations occurred in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the global epidemic still needs further concern. Worryingly, the effectiveness and neutralizing activity of existing antibodies and vaccines against SARS-CoV-2 variants is declining. There is an urgent need to find an effective antiviral medication with broad-spectrum inhibitory effects on novel coronavirus mutant strains against the SARS-CoV-2 infection. Neutralizing antibodies play an important role in the prevention and treatment of COVID-19. The interaction of spike-receptor-binding domain (Spike-RBD) of SARS-CoV-2 and human angiotensin-converting enzyme 2 (ACE2) is the first and critical step of SARS-CoV-2 infection. Hence, the SARS-CoV-2 Spike-RBD is a hot target for neutralizing antibodies development. Evusheld, the combination of Tixagevimab and Cilgavimab monoclonal antibodies (mAbs) targeting Spike-RBD exhibits neutralizing activity against BA.2.12.1, BA.4 and BA.5, which could be used as pre-exposure prophylaxis against SARS-CoV-2 infection. The nucleocapsid (N) protein is a conservative and high-abundance structural protein of SARS-CoV-2. The nCoV396 monoclonal antibody, isolated from the blood of convalescent COVID-19 patients against the N protein of SARS-CoV-2. This mAb not only showed neutralizing activity but also inhibits hyperactivation of complement and lung injury induced by N protein. The mAb 3E8 targeting ACE2 showed broadly neutralizing activity against SARS-CoV-2 and D614G, B.1.1.7, B.1.351, B.1.617.1 and P.1 variants in vitro and in vivo, but did not impact the biological activity of ACE2. Compared with neutralizing antibodies, small molecule inhibitors have several advantages, such as broad-spectrum inhibitory effect, low cost, and simple administration methods. Several small-molecule inhibitors disrupt viral binding by targeting the ACE2 and N-terminal domain (NTD) of SARS-CoV-2 spike protein. Known drugs such as chloroquine and hydroxychloroquine could also block the infection of SARS-CoV-2 by interacting with residue Lys353 in the peptidase domain of ACE2. The transmembrane protease serine 2 (TMPRSS2) inhibitors Camostat mesylate and Proxalutamide inhibit infection by blocking TMPRSS2 mediates viral membrane fusion. The main protease inhibitor Paxlovid and RNA-dependent RNA polymerase inhibitor Azvudine have been approved for treatment of COVID-19 patients. This review summarizes the current research status of neutralizing antibodies and small molecule inhibitors and prospects for their application. We expect to provide more valuable information for further studies in this field.
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@#ObjectiveTo develop and apply a method for detecting the titer of varicella-zoster virus(VZV)neutralizing antibodies based on complement dependence,so as to improve the sensitivity of traditional plaque reduction neutralization assay for detection of the titer of VZV antibody.MethodsThe antigen(live attenuated varicella vaccine)and antibody(human VZV immunoglobulin)were mixed in different proportions and different incubation times. After neutralization,the antigen-antibody mixture was inoculated into human diploid cell 2BS strain cultured in a six-well plate. After 7 ～ 10 d of culture,the number of plaques was counted by Coomassie brilliant blue staining,and the 50% neutralizing antibody titer was calculated by Karber′s formula. Under the optimal neutralization conditions obtained,the effect of complement on the sensitivity of neutralization experiment was explored by changing the addition amount of complement(lyophilized guinea pig serum)to evaluate the optimal addition amount of complement. According to the determined neutralization test parameters,the neutralizing antibody titers of 12 anti-VZV mouse sera and 14 anti-VZV human sera were detected by using traditional plaque method and complement-dependent plaque method respectively.ResultsThe key parameters of the detection method were determined:the titer of VZV standard antigen was 500 ～ 1 000 PFU/mL;the proportion of complement added to the antigen-antibody neutralization system was 1∶10(v/v),and the neutralization condition was 37 ℃ for 1 h. Both the complement-dependent plaque method and the traditional plaque method were positive for anti-VZV mouse serum antibody,while the antibody titer detected by the traditional plaque method was generally lower,and the antibody level of mice inoculated with 2 doses of live attenuated varicella vaccine was significantly higher than that of mice inoculated with 1 dose(t = 0. 45,P < 0. 05);Both of the two methods were positive for anti-VZV human serum antibody.ConclusionA complement-dependent detection method for neutralizing antibody titer of VZV was established. The addition of complement significantly improved the sensitivity of neutralization detection. The evaluation of the titers of neutralizing antibodies in mouse serum with different immunization strategies by the method suggested that the immune effect of two doses of vaccine was better than that of one dose.
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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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So far,the coronavirus disease 2019(COVID-19)has been persisting for nearly three years,infecting about 700 million people and causing more than 6 million deaths,which has seriously affected the human society.According to Global Initiative on Sharing All Influenza Data,there are more than 12 million SARS-CoV-2 variants,of which the five major variants of concern are Alpha,Beta,Gamma,Delta and Omicron.Their infectivity,pathogencity,and neutralization resistance have changed greatly compared with the original strain,which has brought great pressure to the prevention and control of the pandemic.Antibody level testing is critical for confirming infection,epidemiological investigation,vaccine development,and neutralizing drug preparation.Focusing on the humoral immunity against SARS-CoV-2,this paper introduces the mutation sites,neutralization resistance,and vaccination efficacy of the five variants of concern,and briefly summarizes the evolutionary characteristics,future mutation directions,and host immunity.
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Humanos , SARS-CoV-2/genética , Formação de Anticorpos , COVID-19 , Raios gama , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
Emerging SARS-CoV-2 variants have made COVID-19 convalescents susceptible to re-infection and have raised concern about the efficacy of inactivated vaccination in neutralization against emerging variants and antigen-specific B cell response. To this end, a study on a long-term cohort of 208 participants who have recovered from COVID-19 was conducted, and the participants were followed up at 3.3 (Visit 1), 9.2 (Visit 2), and 18.5 (Visit 3) months after SARS-CoV-2 infection. They were classified into three groups (no-vaccination (n = 54), one-dose (n = 62), and two-dose (n = 92) groups) on the basis of the administration of inactivated vaccination. The neutralizing antibody (NAb) titers against the wild-type virus continued to decrease in the no-vaccination group, but they rose significantly in the one-dose and two-dose groups, with the highest NAb titers being observed in the two-dose group at Visit 3. The NAb titers against the Delta variant for the no-vaccination, one-dose, and two-dose groups decreased by 3.3, 1.9, and 2.3 folds relative to the wild-type virus, respectively, and those against the Omicron variant decreased by 7.0, 4.0, and 3.8 folds, respectively. Similarly, the responses of SARS-CoV-2 RBD-specific B cells and memory B cells were boosted by the second vaccine dose. Results showed that the convalescents benefited from the administration of the inactivated vaccine (one or two doses), which enhanced neutralization against highly mutated SARS-CoV-2 variants and memory B cell responses. Two doses of inactivated vaccine among COVID-19 convalescents are therefore recommended for the prevention of the COVID-19 pandemic, and vaccination guidelines and policies need to be updated.
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@#Objective To prepare high titer specific immune serum of varicella-herpes zoster virus(VZV)for the quality control of live attenuated varicella vaccine and live attenuated herpes zoster vaccine.MethodsMale rabbits were immunized with high purity recombinant gE glycoprotein combined with Freund's adjuvant,aluminum hydroxide adjuvant or MF59 adjuvant,2 rabbits in each group. On the 56th day after immunization,the maximum blood samples(heart or carotid artery)were collected from each rabbit to prepare serum,which was mixed with VZV for neutralization reaction,and then inoculated into a 6-well plate full of monolayer of MRC-5 human diploid cells. After incubation for 7 d,the number of plaques was counted and the neutralizing titer and virus neutralizing ability of immune serumwere determined. The serum with high neutralizing titer and virus neutralizing ability was selected for the identification test of live attenuated varicella vaccine and live attenuated herpes zoster vaccine VZV(Oka strain)working seed lot and the detection of exogenous virus factors.ResultsThe immune sera prepared by immunizing rabbits with various combinations of recombinant gE glycoprotein all showed neutralizing activity,among which the serum prepared by the combination of recombinant gE glycoprotein and Freund's adjuvant had the highest neutralizing titer of 1∶512 and the virus neutralizing ability of 240 000 PFU/mL;The prepared immune serum was usedfor the identification test of VZV(Oka strain)working seed lot and the detection of exogenous virus factors,of which all the results were in line with the requirements. Conclusion The recombinantgE glycoprotein could be used for the preparation of high titer neutralizing antibody against VZV,and the prepared high titer neutralizing antibody is suitable for thequality control of live attenuated varicella vaccine and live attenuated herpes zoster vaccine.
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Since an outbreak of coronavirus disease 2019, there have been more than 664 million confirmed cases and more than 6.7 million deaths worldwide.The Omicron variant discovered in November 2021 has become a dominant variant in China.Compared with the general population, Omicron infection mainly presents short-term upper respiratory symptoms followed by quick recovery; solid organ transplant recipients have the characteristics of a high incidence of severe disease and high mortality after infection, and the incidence of secondary infection and rebound positivity is significantly higher than that of the general population.This review focused upon preventive strategies of solid organ transplant recipients from the perspectives of the characteristics of organ transplant recipients, general preventive strategies, vaccination and long-acting neutralizing antibodies.
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Objective:To reduce the immunogenicity of vaccinia virus vector by replacing the D8L region, which is a neutralizing antibody epitope in vaccinia virus, with an exogenous gene.Methods:A gene fragment encoding influenza virus hemagglutinin (HA) was inserted into the D8L region to replace it using homologous recombination technique. Then, a recombinant vaccinia virus influenza vaccine was constricted. A recombinant vaccinia virus vaccine with the TK region expressing HA was used as a control. The expression of HA was validated by Western blot. BALB/c mice were immunized with the vaccines and the serum antibody titers two weeks after each immunization were evaluated by ELISA and hemagglutination inhibition assay. The protective efficacy of the recombinant vaccinia virus was assessed through a challenge experiment.Results:Western blot confirmed the successful expression of HAD8L protein in the constructed recombinant vaccines. ELISA and hemagglutination inhibition assay showed that after the primary immunization, the anti-HA antibody titer induced by the recombinant vaccinia virus with D8L region mutation was slightly higher than that induced by the vaccine with TK region mutation, and the difference was statistically significant with the increase of immunization times ( P<0.05). The recombinant vaccinia virus with D8L region mutation showed significantly lower immunogenicity than the recombinant virus with TK region mutation after the primary immunization, but there was no significant difference between them with the increase of immunization times ( P>0.05). After H1N1pdm challenge, no virus was detected in the mice immunized with the recombinant vaccinia virus with D8L region mutation and the mice showed mild lung inflammation and less tissue damage. Conclusions:This study indicated that inserting exogenous genes into the D8L region of the neutralizing antibody epitope in the vaccinia virus vector could help to reduce the immunogenicity of the vector itself and enhance the immunogenicity of the exogenous genes. This provided a reference for the use of the vaccinia virus vector as a delivery tool in the field of vaccines or gene therapy.
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Influenza is a worldwide infectious disease caused by influenza virus. It has posed great challenges on public health and social stability since 1918. At present, vaccination is the most effective way to prevent and control influenza epidemics. Broad-spectrum antiviral drugs and neutralizing antibodies against influenza virus have been widely studied in recent years. Hemagglutinin (HA), which is on the surface of influenza virus, plays an important role in the stage of viral invasion into host cells. It is the main effective antigenic component of current influenza vaccines, as well as the main target of broad-spectrum neutralizing antibodies and broad-spectrum antiviral drugs. This review summarized the progress in the development of novel influenza vaccines, neutralizing antibodies, and antiviral drugs based on influenza virus HA, as well as other prevention and control measures, hoping to present new ideas for future influenza prevention and control.
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ObjectiveTo determine the difference of serum neutralizing antibody levels in healthy persons following the vaccination of inactivated SARS-CoV-2 vaccines. MethodsHealth care workers that received inactivated SARS-CoV-2 vaccines were enrolled in Sichuan provincial people's hospital from January to December 2021. All participants were classified into four groups according to the number and time of vaccination， which were groups of 28 days after the second dose， 180 days after the second dose， 28 days after the third dose and 150 days after the third dose. Serum neutralizing antibody was quantitatively detected by chemiluminescence method. Furthermore， the serum neutralizing antibody levels were compared within and between groups by gender， age and body mass index（BMI）. ResultsA total of 349 participants were enrolled in this study， including 113 males and 236 females. The positive rates of neutralizing antibody in the groups of 28 days after the second dose， 180 days after the second dose， 28 days after the third dose and 150 days after the third dose were 74.0%， 25.7%， 100.0% and 100.0%， respectively. In the four groups， neutralizing antibody levels were 10.38 （5.76， 24.00） AU·mL-1， 4.18 （3.00， 6.18） AU·mL-1， 41.18 （25.80， 116.21） AU·mL-1 and 33.33 （18.09， 69.12） AU·mL-1， respectively. The positive rate of neutralizing antibody significantly differed across the groups （P<0.001）. Furthermore， neutralizing antibody level in the third dose groups were significantly higher than that in the second dose groups （P<0.001）. Neutralization antibody level in young people （<45 years old） was significantly higher than that in middle-aged and elderly people （≥45 years old） in the groups of 180 days after the second dose and 28 days after the third dose （P<0.05）. Additionally， neutralization antibody level in normal weight participants was significantly higher than that in overweight and obese participants in the group of 28 days after the second dose （P<0.05）. However， no significant difference was found in all groups by gender （P>0.05）. ConclusionCompared with two doses of inactivated SARS-CoV-2 vaccine， three doses can significantly induce higher neutralizing antibody and stronger immune protection. Age and BMI have certain effect on the neutralizing antibody.
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【Objective】 To analyze the results of different methods for reactive samples screened by the enzyme linked immunosorbent assay (ELISA) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in blood donors. 【Methods】 From March to April 2020, a total of 8 632 blood samples in Shenzhen were screened for SARS-CoV-2 total antibodies (TAb, including IgG, IgM, IgA) in plasma using ELISA(PC group), the antibody reactivity samples and their follow up plasma samples (FC group), and samples of disease control group(DC group) from January to April 2020 were detected using the following methods: 1) ELISA method for detecting IgG, IgM, and (or without detection) TAb; 2) pseudovirus neutralizing antibody test(pVNT); 3) western blot (WB) of SARS-CoV-2 antibody. The negative control group(NC group) from February to April 2020 performed ELISA and WB testing. 【Results】 Among the 34 total antibody positive samples, 2 were positive for pVNT test, and the total antibody, IgG and WB in the initial screening and tracking testing were positive. Thereafter, it was determined to be confirmed positive. The other 2 cases were positive for pVNT test, while the samples with positive WB results were in the follow-up stage. The TAb, IgG, and pVNT results did not conform to the dynamic evolution of antibodies, and cannot be determined as confirmed positive. 【Conclusion】 The infection status of antibody reactivity samples screened by SARS-CoV-2 ELISA can be judged by the logic of pVNT, WB and the dynamic change of antibody.
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【Objective】 To study the relationship between the plasma IgG, IgM, neutralizing antibody titer and sex, age, collection interval in convalescent patients with COVID-19, so as to guide the plasma collection of convalescent patients with COVID-19. 【Methods】 COVID-19 convalescent plasma was collected to determine the antibody titer, and the difference and correlation of data in each group were analyzed by SPSS statistical analysis software. 【Results】 The median titers (AU/mL)of IgG, IgM and neutralizing antibodies in males and females were 484.24 vs 516.04, 2.13 vs 1.73, and 1 124.74 vs 1 143.99, respectively, and there was no significant difference(P>0.05) . Age had weak positive correlation with IgG and neutralizing antibody, and the Spearman correlation coefficient was 0.188 (P<0.05). The median titers (AU/mL) of IgG, IgM and neutralizing antibody at first donation of 30 repeated donors were 522.3, 2.64 and 1 174.6, respectively, but at second donation were 332.08, 0.63 and 708.96, showing significant difference (P<0.05). 【Conclusion】 There was no significant difference in the plasma IgG, IgM and neutralizing antibody titers in convalescent COVID-19 patients of different ages and genders, and the titers met the requirements of clinical treatment guidelines. Although the plasma antibody level of repeated donors has decreased, it still has clinical value.
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【Objective】 To determine the best collection time period of plasma which can be used for human COVID-19 immunoglobulin for intravenous injection through SARS-CoV-2-IgG change and neutralizing antibody distribution against different virus strain in representative mixed plasma before and after Omicron strain infection by ELISA and pseudovirus neutralization test. 【Methods】 An ELISA method for quantitative detection of SARS-CoV-2-IgG was established and its linear range,accuracy and precision was verified. SARS-CoV-2-IgG potency was detected in 25 convalescent plasma which were collected 20-40 days after confirmed Omicron infection, two groups of mixed plasma samples WP1 and WP2 were prepared according to the SARS-CoV-2-IgG results, and pseudovirus neutralization experiments with different virus strain (prototype strain, BA. 1,BA.2, BA.4/5, BF.7, BQ.1.1) were carried out to determine the distribution of neutralizing antibodies against different virus strain. SARS-CoV-2-IgG potency of representative mixed plasma collected from 14 plasma stations subordinate to the company before and after Omicron strain infection was detected, including Omicron convalescent plasma (OP) collected from different plasma stations from December 2022 to May 2023 and normal pool plasma (VN) feed in March 2023 which collected from March 2022 to December 2022. According to the results, the difference and the change rule with time of SARS-CoV-2-IgG before and after Omicron strain infection were analyzed. 【Results】 The linearity of SARS-CoV-2-IgG ranged from 6.25 to 200 EIU/mL, the accuracy in-batch ranged from 81.793% to 106.985%, the precision in-batch ranged from 1. 100% to 13.000%, and the total error in-batch ranged from 2.988% to 22.679%. The accuracy between batches ranged from 90.788%to 96.893%, the precision between batches ranged from 4.870% to 6.272%, and the total error between batches ranged from 9.192% to 15.399%. The results of pseudovirus neutralizing antibody showed that the potency of different virus strain neutralizing antibodies were in the order of prototype strain>BA.2>BA.4/5>BF.7≈ BQ.1.1>BA.1 and the correlation between WP1 and WP2 was high (Pearson r=0. 931 1, P=0.002 3) which indicated that the potency distribution of neutralizing antibodies of different virus strain in Omicron convalescent plasma was basically stable. Compared with the mixed convalescent plasma sample G128 collected in June 2022, the potency of Omicron neutralizing antibodies of WP series were significantly higher, the ratio of BA.2 antibody to prototype antibody increased from 26.9% (before infection) to 82.6%-87.5% (after infection). The results of VN series before Omicron infection were < 100 EIU/mL, and the results of OP series after Omicron infection showed that the plasma collected from the beginning of December 2022 was the peak of antibody in the same month,and then dropped sharply, entering a short plateau in February-March 2023 (potency was about 40% of the peak value),and then dropped sharply again in April (potency was about 20% of the peak value). 【Conclusion】 The potency and proportion of neutralizing antibody against Omicron subtype in convalescent plasma after COVID-19 Omicron strain infection increased significantly. IgG antibody of plasma donors in different regions reached its peak in the month of infection, then continued to dropped sharply. The best collection period of plasma that can be used for human COVID-19 immunoglobulin for intravenous injection was 1 to 2 months after infection.
RESUMO
【Objective】 To investigate the trend of neutralizing antibody level in plasma donors who received the 3rd shot of inactivated novel coronavirus vaccine. 【Methods】 Three commercial ELISA kits for novel coronavirus neutralization antibody detection, manufactured by Company A, B and C, were chosen and screened by Pseudotype Neutralization Test from December 2021 to June 2022. A total of 410 plasma samples from 64 plasma donors who received the 3rd shot of inactivated novel coronavirus vaccine and there after donated plasma within six months were detected by the selected ELISA kit from July to October, 2022. The data were analyzed by Excel 2013 and SPSS 26 software. 【Results】 The high-throughput ELISA kit for SARS-CoV-2 neutralizing antibody detection, manufactured by Company A, was selected for further antibody titer detection. The mixed plasma titers were 1 337.34, 1 148.89, 852.19, 681.38, 556.44 and 457.19 U/mL from 1 to 6 months, respectively, after the 3rd shot of vaccine. The neutralizing antibody titer level began to increase around 7 days after the 3rd shot of vaccine injection and peaked (peak range: 264.07-2 208.39 U/mL, median: 569.34 U/mL) at 1 month (range: 9-43 days, median: 22 days), and then gradually decreased (P<0.05). 【Conclusion】 The neutralizing antibody titer of plasma donors who received the 3rd shot of inactivated novel coronavirus vaccine began to rise around 7 days after vaccination, which reached the peak value at around 1 month and then gradually decreased.