血清抗百日咳毒素中和抗体检测方法的建立及应用

目的 建立血清抗百日咳毒素(pertussis toxin,PT)中和抗体的检测方法,并进行应用.方法 将不同浓度的PT蛋白(0.1～105.5 ng/mL)和105个/mL的CHO细胞悬浮液加入96孔板,均100 μL/孔,37℃孵育24 h,显微镜下观察细胞簇集程度,确定中和试验中PT的最适终浓度.将待检血清(1:...     

抗狂犬病病毒抗体的制备及酶联免疫法的建立

目的制备具有中和活性的抗狂犬病病毒(Rabiesvirus,RV)抗体,并建立检测RV抗原的ELISA法。方法以RV全病毒免疫2只新西兰家兔,制备抗RV多克隆抗体。以RV全病毒免疫BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0融合,建立稳定分泌抗RV单克隆抗体的杂交瘤细胞株。以小鼠中和试验(MNT)检测抗体的中和活性。以ELISA双抗体夹心法和ELISA竞争法检测RV抗原。结果2只家兔的多克隆抗体ELISA效价分别为1∶6.0×103和1∶1.2×104,纯化的兔抗RVIgG中和活性分别为46.3和29.2IU/ml。获得了9株稳定分泌抗RV的单克隆抗体杂交瘤细胞株,属于IgG1或IgG2b亚型,腹水的抗体ELISA效价为1∶1.0×105~1∶1.0×107。单克隆抗体3E5、4C2和4F8具有中和活性,Westernblot分析提示,单克隆抗体4C2是针对RV糖蛋白线性表位的抗体。以单克隆抗体4C2作为捕捉抗体,兔多克隆抗体作为检测抗体,建立了ELISA双抗体夹心法,检测RV抗原。同时建立了另一种ELISA竞争法,加入固定工作浓度的单克隆抗体4C2,与RV病毒液孵育,以ELISA间接法检测RV病毒抗原。结论所获得的狂犬病毒多克隆和单克隆抗体具有中和活性,可在ELISA中用于检测RV抗原。     

柯萨奇病毒A组16型VP1蛋白的原核表达及其免疫原性

目的原核表达柯萨奇病毒A组16型(Coxsackievirus group A type 16,CA16)VP1蛋白,并检测其免疫原性。方法通过RT-PCR法从CA16病毒青岛株中扩增VP1基因,克隆至原核表达载体pET43.1a(+)中,构建重组表达质粒pET43.1a-VP1,转化感受态E.coli Rossatte(DE3),IPTG诱导表达。表达的重组CA16 VP1蛋白通过Ni柱亲和层析纯化后,采用不同剂量(5、10、20、40μg)免疫BALB/c小鼠,ELISA法检测血清中特异性IgG、IgG1、IgG2a、IgG2b、IgG3抗体效价,微量细胞病变抑制法检测血清中和抗体效价。结果重组表达质粒pET43.1a-VP1经双酶切及测序证实构建正确;表达的重组CA16 VP1蛋白相对分子质量约为34 000,主要以包涵体形式存在,表达量占菌体总蛋白的15%;纯化的重组CA16 VP1蛋白纯度可达95%以上,可与猴CA16抗血清反应;不同剂量的重组CA16 VP1蛋白免疫BALB/c小鼠,可诱导产生CA16特异性抗体,血清中总IgG、IgG1、IgG2a、IgG2b、IgG3抗体效价均明显高于对照组,且抗体效价与免疫剂量存在一定的量效关系;各剂量CA16 VP1组免疫小鼠血清中和抗体效价均小于1∶8。结论已成功在大肠杆菌中表达了重组CA16 VP1蛋白,纯化的重组蛋白可诱导小鼠特异性体液免疫应答,为进一步研究CA16的结构、功能及相关疫苗的研制奠定了基础。     

Self-Assembled Particles Combining SARS-CoV-2 RBD Protein and RBD DNA Vaccine Induce Synergistic Enhancement of the Humoral Response in Mice

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.     

Identification of Entry Inhibitors against Delta and Omicron Variants of SARS-CoV-2

Entry inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to control the outbreak of coronavirus disease 2019 (COVID-19). This study developed a robust and straightforward assay that detected the molecular interaction between the receptor-binding domain (RBD) of viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor in just 10 min. A drug library of 1068 approved compounds was used to screen for SARS-CoV2 entry inhibition, and 9 active drugs were identified as specific pseudovirus entry inhibitors. A plaque reduction neutralization test using authentic SARS-CoV-2 virus in Vero E6 cells confirmed that 2 of these drugs (Etravirine and Dolutegravir) significantly inhibited the infection of SARS-CoV-2. With molecular docking, we showed that both Etravirine and Dolutegravir are preferentially bound to primary ACE2-interacting residues on the RBD domain, implying that these two drug blocks may prohibit the viral attachment of SARS-CoV-2. We compared the neutralizing activities of these entry inhibitors against different pseudoviruses carrying spike proteins from alpha, beta, gamma, and delta variants. Both Etravirine and Dolutegravir showed similar neutralizing activities against different variants, with EC50 values between 4.5 to 5.8 nM for Etravirine and 10.2 to 22.9 nM for Dolutegravir. These data implied that Etravirine and Dolutegravir may serve as general spike inhibitors against dominant viral variants of SARS-CoV-2.     

戊型肝炎病毒重组蛋白P179抗原单克隆抗体及抗原检测试剂盒的制备

目的制备戊型肝炎病毒(Hepatitis E virus,HEV)重组蛋白P179抗原单克隆抗体及抗原检测试剂盒。方法以4型HEV ORF2重组蛋白P179免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,间接ELISA法筛选阳性杂交瘤细胞株,有限稀释法进行克隆化培养,制备腹水,并对杂交瘤细胞进行染色体计数,鉴定单抗亚类及特异性、并检测稳定性;经SDS-PAGE分析抗体纯度,以兔抗179抗原多抗作为包被抗体,HRP标记P179单抗作为酶标抗体,建立双抗体夹心ELISA法,制备试剂盒,并进行最佳线性范围测定、准确性、精密性和稳定性验证。结果获得4株能稳定分泌抗P179抗原单抗的杂交瘤细胞株,分别命名为3A10、3F8、4E9和5G6,腹水抗体效价分别为1∶10-7、1∶10-6、1∶10-6和1∶10-7,染色体数分别为96、98、101和99条;亚类分别为IgG2a、IgG2a、IgG1和IgG2b;4株单抗连续传代3个月,液氮冻存1年抗体效价未发生变化;制备的试剂盒有良好的线性(R2>0.950 0)、准确性、精密性,试验内变异系数为4.17%～6.26%,回收率在87.9%～114.8%之间,试验间变异系数为5.82%～8.01%,回收率在90.8%～108.9%之间;于37℃及4℃放置3 d,仍具有良好的稳定性。结论成功制备了戊型肝炎病毒P179抗原单克隆抗体及抗原检测试剂盒,可用于戊型肝炎疫苗生产中定量检测疫苗抗原。     

抗肿瘤坏死因子相关凋亡诱导配体细胞外段单克隆抗体的制备

目的制备抗肿瘤坏死因子相关凋亡诱导配体细胞外段(sTRAIL)的单克隆抗体。方法用重组sTRAIL免疫BALB/c小鼠,取脾细胞与SP2/0细胞融合,捕获ELISA筛选阳性克隆,制备腹水抗体。间接ELISA测定单抗效价,并进行单抗亚类、特异性鉴定及杂交瘤细胞染色体和单抗识别位点分析。将杂交瘤细胞体外连续培养3个月及冻存6个月后,检测细胞培养上清的效价。结果3株杂交瘤细胞分泌的单抗均为IgG1型,腹水单抗效价均高于10-6,杂交瘤细胞染色体数介于94～98条之间,均识别sTRAIL分子上线性表位。细胞体外连续培养3个月及冻存6个月后的上清效价保持稳定。结论已成功制备了3株可稳定分泌抗sTRAIL单克隆抗体的杂交瘤细胞,为TRAIL的定性定量检测及组织表达分析奠定了基础。     

猪血凝性脑脊髓炎病毒单克隆抗体的制备与鉴定

目的制备猪血凝性脑脊髓炎病毒(HEV)单克隆抗体,并进行鉴定。方法通过差速离心和蔗糖密度梯度离心法对猪HEV进行纯化,免疫BALB/c小鼠后,取其脾细胞与骨髓瘤SP2/0细胞进行融合,经间接ELISA和血凝抑制试验(HI)筛选能稳定分泌抗HEV单克隆抗体的杂交瘤细胞株,并对单抗进行生物学鉴定。结果筛选出4株能稳定分泌抗HEV单抗的杂交瘤细胞株2H2、2A1、1E2、4D4。经鉴定,4株杂交瘤细胞株诱生小鼠腹水抗HEV的ELISA效价可达1∶12800～1∶51200,其中3株HI效价可达1∶24～1∶28,另1株为0。4株杂交瘤细胞分泌的单抗与猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)和猪伪狂犬病病毒(PRV)均不发生交叉反应;杂交瘤细胞染色体数为83～103;除2A1单抗为IgG2b外,其他3株均为IgG1;Westernblot分析表明,2H2、2A1和4D4能识别HEV的血凝素-酯酶蛋白(HE),1E2可识别纤突蛋白(S)。结论已成功制备出抗HEV的单克隆抗体,为HEV快速检测试剂的研制奠定了基础。     

静注甲型H1N1流感人免疫球蛋白的研制

目的研制高效价静注甲型H1N1流感人免疫球蛋白。方法用甲型H1N1流感疫苗(简称甲流)对固定供血浆者按疫苗说明书免疫后2周开始采集原料血浆,采用血凝抑制法检测原料血浆和制品的甲流抗体效价;采用柱层析法从高效价甲流免疫血浆中提取富含IgM免疫球蛋白的样品,模拟低温乙醇蛋白分离法工艺从小量混合血浆分离IgG样品,测定血浆和提取样品的甲流抗体效价,分析甲流中和抗体效价与IgG、IgM含量的对应关系,判断甲流中和抗体的免疫球蛋白类型;按本公司静脉注射人免疫球蛋白(Intravenous immunoglobulin,IVIG)生产工艺制备静注甲型H1N1流感人免疫球蛋白,并与普通IVIG及相应合并血浆的甲流抗体效价进行比较。结果筛选到甲流抗体效价≥320 HU/ml的合格血浆9 866袋,合格率达33.5%,其中抗体效价≥640 HU/ml的血浆占合格血浆的49%,血浆质量符合《中国药典》三部(2010版)"血液制品生产用人血浆规程"要求;甲流抗体效价与IgG含量呈相应比例关系,而与IgM含量无关,甲流中和抗体的免疫球蛋白类型以IgG为主;制备的甲流人免疫球蛋白制品的甲流中和抗体效价达2 560 HU/ml,为普通IVIG的197倍,其他质量指标符合《中国药典》三部(2010版)"静注人免疫球蛋白制检规程"要求。结论成功研制了静注甲型H1N1流感人免疫球蛋白,在甲流疫情得到有效控制的情况下,仍具有战略储备意义。     

高密度介质固相抗人球蛋白法检测孕妇ABO血型系统IgG抗体效价

目的探讨利用高密度介质固相抗人球蛋白法检测孕妇ABO血型系统IgG抗体效价的可行性。方法应用不完全抗体检测试剂盒(高密度介质固相抗人球蛋白法)检测40例O型血孕妇ABO血型系统IgG抗体效价,并与传统抗人球蛋白试管法进行对比。结果以IgG抗A(B)效价≥1:64定为阳性,两种方法的阳性检出率一致,均为50%(20/40);两种方法检测的抗体效价的几何均数差异无统计学意义(P>0.05);两种方法高度相关,r=0.9079(tr=13.36,P<0.001)。结论高密度介质固相抗人球蛋白法与传统抗人球蛋白试管法比较,操作简单,耗时短,结果准确,可应用于ABO血型系统IgG抗体效价的检测。     

巨细胞病毒IgG定量ELISA检测方法的建立及初步应用

目的建立巨细胞病毒(Cytomegalovirus,CMV)IgG定量ELISA检测方法,并进行初步应用。方法用CMV IgG(50 U/ml)标准品建立CMV IgG ELISA定量检测方法,确定该方法的最佳线性范围;制备室内质控血清并进行标定。对该方法进行重复性、中间精密性、准确性、特异性验证,并与Dia.Pro CMV IgG ELISA试剂盒进行比较。用建立的ELISA法检测689人份健康献浆员血浆的CMV IgG抗体效价。结果建立的CMV IgG定量ELISA检测方法的最佳线性范围为0.000 78～0.05 U/ml;制备的室内质控血清的抗体效价为(5.67±0.55)U/ml;重复性试验检测结果的变异系数为4.6%～9.8%,中间精密性试验检测结果的变异系数为9.7%～10.6%;该方法检测3个浓度标准品的回收率为107.01%～112.97%;样品稀释液和血清中的其他一些干扰因素对检测结果影响较小;该方法与Dia.Pro CMV IgG ELISA试剂盒检测血浆样品的相关系数r=0.81。该方法检测689人份健康献浆员血浆样品的CMV IgG抗体效价≥10 U/ml的占30.8%。结论建立的CMV IgG定量ELISA检测方法操作简便,精密性良好,特异性强,可用于人血浆中CMV IgG的定量检测。     

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell’s receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.     

抗肠道病毒71型单克隆抗体的制备及鉴定

目的制备肠道病毒71型(EV71)单克隆抗体,并进行初步鉴定。方法将RD细胞和Vero细胞培养的EV71原液通过氯化铯超速离心纯化,分别作为免疫抗原和检测抗原,制备单克隆抗体。通过Westernblot分析、间接免疫荧光试验和ELISA法鉴定单抗的特异性。采用间接ELISA法测定单抗的酶标效价,微量细胞培养中和试验测定单抗的中和效价。结果获得1株可稳定分泌抗EV71单克隆抗体的杂交瘤细胞株,其分泌的单抗可与EV71特异性结合,酶标效价为1∶204800,中和效价为1∶28。结论已成功筛选出1株分泌抗EV71的单抗细胞株,为建立EV71疫苗抗原含量测定方法奠定了基础。     

Ⅱ型单纯疱疹病毒糖蛋白D在CHO细胞中的表达及其免疫学特性

目的在CHO细胞中表达Ⅱ型单纯疱疹病毒(Herpes simplex virus-2,HSV-2)糖蛋白D(Glycoprotein D,gD),并检测其免疫学特性。方法采用Vero细胞培养HSV-2,提取总DNA,以其为模板,PCR扩增gD基因,与pCMV-sport载体连接,构建重组表达质粒pCMV-gD,将质粒pCMV-gD转染COS-7和CHO-K1细胞,并进行表达。亲和胶Anti-flag M2 Agarose纯化表达蛋白,经SDS-PAGE和HPLC分析重组蛋白纯度,Western blot分析蛋白反应原性,全波长扫描分析蛋白的光谱曲线,等电聚焦电泳测定蛋白的等电点。以20、40μg纯化的gD免疫BALB/c小鼠,ELISA法检测小鼠血清中HSV-2 gD特异性抗体水平,中和试验测定小鼠血清中和抗体水平。结果酶切鉴定及DNA测序表明,重组表达质粒pCMV-gD构建正确,在COS-7细胞的瞬时表达产物和CHO细胞中的稳定表达产物均可被HSV-2 gD单抗特异性识别,表明该蛋白具有较好的反应原性。纯化的gD在相对分子质量约5 500处可见目的蛋白条带,纯度为65.46%;保留天然HSV-2 gD的反应原性,最适紫外吸收波长为275.50 nm,等电点为8.3。gD 20μg组和40μg组小鼠血清特异性抗体滴度分别为1∶125 000和1∶16 000,中和抗体滴度分别为1∶17和1∶16,表明gD可诱导中和抗体的产生,也可诱导高滴度的HSV-2gD特异性抗体。结论成功在CHO细胞中稳定表达了HSV-2 gD,表达的HSV-2 gD具有较好的免疫原性,为基因工程疫苗的开发奠定了基础。     

人呼吸道合胞病毒与结核分枝杆菌融合蛋白TB10.4-F1的免疫原性

目的在大肠杆菌中表达并纯化人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)和结核分枝杆菌(Mycobacterium tuberculosis,MTB)的融合蛋白TB10.4-F1,检测其免疫原性。方法将重组工程菌TB10.4/pET28a/BL21和TB10.4-F1/pET30a/BL21经IPTG诱导表达,SDS-PAGE分析蛋白的表达形式;表达的融合蛋白TB10.4和TB10.4-F1经His TrapTMHP层析柱进行亲和层析一步纯化后,免疫BALB/c小鼠。小鼠分为TB10.4(30μg/只)、TB10.4-F1(30μg/只)及PBS(200μl)组,分别于0、2、4周免疫小鼠,于每次免疫前1 d经尾部取血,分离血清,ELISA法检测小鼠血清中特异性的IgG水平;于末次免疫2周后摘眼球取血,分离血清,ELISA法检测特异性IgG水平及中和抗体滴度。结果表达的重组融合蛋白TB10.4和TB10.4-F1的相对分子质量分别约13 300和59 600,均以包涵体形式表达,表达量分别占菌体总蛋白的53.9%和12%;纯化后的重组融合蛋白TB10.4和TB10.4-F1纯度分别可达95%和80%;与TB10.4组相比,TB10.4-F1组IgG水平明显升高,IgG1/IgG2a值明显降低,但仍大于1;TB10.4-F1组小鼠血清中RSV特异性中和抗体滴度可达log102.699。结论融合蛋白TB10.4-F1免疫原性强,能激发出较为平衡的Th1/Th2反应,并产生抗HRSV的中和抗体。融合蛋白TB10.4-F1有望成为预防HRSV感染的候选疫苗。     

肠道病毒71型中和抗体的保护水平

目的通过检测乳鼠模型、健康婴幼儿和疫苗免疫后目标人群的肠道病毒71型(Enterovirus 71,EV71)中和抗体效价,为确定EV71疫苗保护水平提供基础数据。方法 6份不同EV71病毒株免疫血清经中和抗体准确定量后,分别通过两个EV71乳鼠攻毒模型,检测EV71中和抗体保护水平。采用标准化的中和抗体检测法和EV71中和抗体定值标准品,分别对健康婴幼儿及疫苗免疫后血清进行中和抗体准确定量。结果在2个乳鼠攻毒模型中,6份免疫血清的中和抗体保护水平接近,ED50分别为10～50 U和6.1～54.2 U;婴幼儿人群7、12和24月龄抗体阳性率分别为45.0%、48.8%和56.8%,阳性人群的抗体效价分别为113.8、120.3和330.6U/m(l均值为173.2 U/m)l,母亲抗体阳性率和效价分别为86.7%和114.3 U/ml;中、高剂量疫苗2针免疫后,婴幼儿抗体阳性率均为100%,效价为356.0和1 136.2 U/ml,较婴幼儿人群自然感染水平高2.1～6.6倍。结论应用标准化的中和抗体检测法和EV71中和抗体定值标准品,确定了以U/ml为单位的乳鼠攻毒抗体保护水平及婴幼儿人群抗体水平和疫苗免疫后抗体水平,为EV71疫苗免疫人群中和抗体保护水平的确定提供了依据。     

人巨细胞病毒gB基因真核表达载体在小鼠中的免疫效果

目的观察人巨细胞病毒(HCMV)gB基因真核表达载体在小鼠中的免疫效果。方法大量提取目的质粒,分3个剂量组进行多点肌肉注射免疫小鼠,利用MTT法检测免疫小鼠的T细胞增殖活性,ELISA法检测小鼠血清中HCMV特异性抗体,中和试验检测小鼠血清中和抗体。结果ELISA检测结果表明,与空白对照、空载体对照组相比,3个剂量组pcD-NA3.1/gB质粒免疫后小鼠血浆中抗HCMV IgG抗体水平均明显增高(P<0.05),中和抗体水平为1∶20~1∶40,而对照组血清中无中和抗体存在。淋巴细胞增殖指数免疫小鼠与正常小鼠差异无显著意义。结论已构建的HCMV gB基因真核表达载体可以诱导小鼠产生明显的体液免疫,为核酸疫苗研究奠定了基础。     

SARS-CoV-2 Variants,RBD Mutations,Binding Affinity,and Antibody Escape

Since 2020, the receptor-binding domain (RBD) of the spike protein of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been constantly mutating, producing most of the notable missense mutations in the context of “variants of concern”, probably in response to the vaccine-driven alteration of immune profiles of the human population. The Delta variant, in particular, has become the most prevalent variant of the epidemic, and it is spreading in countries with the highest vaccination rates, causing the world to face the risk of a new wave of the contagion. Understanding the physical mechanism responsible for the mutation-induced changes in the RBD’s binding affinity, its transmissibility, and its capacity to escape vaccine-induced immunity is the “urgent challenge” in the development of preventive measures, vaccines, and therapeutic antibodies against the coronavirus disease 2019 (COVID-19) pandemic. In this study, entropy–enthalpy compensation and the Gibbs free energy change were used to analyze the impact of the RBD mutations on the binding affinity of SARS-CoV-2 variants with the receptor angiotensin converting enzyme 2 (ACE2) and existing antibodies. Through the analysis, we found that the existing mutations have already covered almost all possible detrimental mutations that could result in an increase of transmissibility, and that a possible mutation in amino-acid position 498 of the RBD can potentially enhance its binding affinity. A new calculation method for the binding energies of protein–protein complexes is proposed based on the entropy–enthalpy compensation rule. All known structures of RBD–antibody complexes and the RBD–ACE2 complex comply with the entropy–enthalpy compensation rule in providing the driving force behind the spontaneous protein–protein docking. The variant-induced risk of breakthrough infections in vaccinated people is attributed to the L452R mutation’s reduction of the binding affinity of many antibodies. Mutations reversing the hydrophobic or hydrophilic performance of residues in the spike RBD potentially cause breakthrough infections of coronaviruses due to the changes in geometric complementarity in the entropy–enthalpy compensations between antibodies and the virus at the binding sites.     

Q fever endocarditis in Romania: the first cases confirmed by direct sequencing

Infective endocarditis (IE) is a serious, life-threatening disease with highly variable clinical signs, making its diagnostic a real challenge. A diagnosis is readily made if blood cultures are positive, but in 2.5 to 31% of all infective endocarditis cases, routine blood cultures are negative. In such situations, alternative diagnostic approaches are necessary. Coxiella burnetii and Bartonella spp. are the etiological agents of blood culture-negative endocarditis (BCNE) most frequently identified by serology. The purpose of this study is to investigate the usefulness of molecular assays, as complementary methods to the conventional serologic methods for the rapid confirmatory diagnostic of Q fever endocarditis in patients with BCNE. Currently, detection of C. burnetii by culture or an antiphase I IgG antibody titers >800 represents a major Duke criterion for defining IE, while a titers of >800 for IgG antibodies to either B. henselae or B. quintana is used for the diagnosis of endocarditis due to Bartonella spp. We used indirect immunofluorescence assays for the detection of IgG titers for C. burnetii, B. henselae and B. quintana in 57 serum samples from patients with clinical suspicion of IE. Thirty three samples originated from BCNE patients, whereas 24 were tested before obtaining the blood cultures results, which finally were positive. The results of serologic testing showed that nine out of 33 BCNE cases exhibited antiphase I C. burnetii IgG antibody titer >800, whereas none has IgG for B. henselae or B. quintana. Subsequently, we used nested-PCR assay for the amplification of C. burnetii DNA in the nine positive serum samples, and we obtained positive PCR results for all analyzed cases. Afterwards we used the DNA sequencing of amplicons for the repetitive element associated to htpAB gene to confirm the results of nested-PCR. The results of sequencing allowed us to confirm that C. burnetii is the causative microorganism responsible for BCNE. In conclusion, the nested PCR amplification followed by direct sequencing is a reliable and accurate method when applied to serum samples, and it may be used as an additional test to the serological methods for the confirmatory diagnosis of BCNE cases determined by C. burnetii.